Long emission wavelength chemiluminescent compounds and their use in test assays

ABSTRACT

An assay method incorporating at least two different chemiluminescent compounds for detection and/or quantitation of at least two substances in a test sample is described. The synthesis of chemiluminescent reagents or conjugates for use in such methods as well as kits incorporating such reagents are also disclosed. The assays have particular application in the field of clinical diagnostics.

This is a divisional of application Ser. No. 08/035,130 filed on Mar.19, 1993, U.S. Pat. No. 5,395,752.

BACKGROUND OF THE INVENTION 1. TECHNICAL FIELD

The present invention relates to a new class of chemiluminescent,aromatic ring-fused acridinium compounds (AFAC) which emit green oryellow light upon simple chemical treatments. This invention alsorelates to conjugates formed from AFAC and binding partners, e.g.biological molecules, and test assays utilizing the conjugates.Furthermore the invention relates to test assays in which the detectionand/or quantitation of two or more substances or analytes in a testsample can be carried out simultaneously due to the discernable andnon-interfering light emission characteristics of two or morechemiluminescent conjugates.

2. CROSS-REFERENCE

The following application, filed concurrently herewith, describes aluminometer for detecting emission spectra of at least twochemiluminescent compounds in a test sample and is identified as U.S.Ser. No. 08/035,341, abandoned. The disclosure of said application iscommonly assigned and incorporated herein by reference.

3. TECHNICAL REVIEW

Chemiluminescent compounds that emit light with separated wavelengthmaxima and minimally overlapping but correctable emission spectra can bevery useful in analytical assays, particularly in industrial assays, andin clinical diagnostic assays for multi-substance, e.g. multi-analyte,determinations. Such compounds can be used to tag or label bindingpartners, e.g. biological molecules, such as antigens, antibodies, andnucleic acids to form conjugates or tracers that are capable ofproducing mutually non-interfering, or minimally over-lapping lightemission signals or spectra, that allow the simultaneous detectionand/or quantitation of multiple substances in a test sample. Forexample, simultaneous determinations of serum levels of luteinizinghormone (LH) and follicle stimulating hormone (FSH) from one patientsample is possible and is demonstrated below, because twochemiluminescent compounds having light emission spectra which spanabout 100-250 nm for a spectral region with signal intensity above 5% ofpeak height, but differing in their emission maxima so that the signalsare discernable. In one example the emission maxim of twochemiluminescent signals differ by about 60 nm and preferably by 80 nmor more for labeling or tagging anti-LH and anti-FSH. A further examplewhere simultaneous determinations is possible according to the methodsdescribed herein, is in the assay of amplified nucleic acid sequences,e.g. oncogenes associated with malignant transformation. See EP-A-0 481704 (priority application U.S. Ser. No. 598,269 (Oct. 16, 1990),abandoned and references cited therein) which is commonly assigned andincorporated herein by reference. In such assays, the inclusion of aparallel, internal reference material for a known, different targetsequence in the same working vessel or reaction medium as a positivecontrol is recognized as important to assay performance, for example, tosafeguard false negative results. In other assay formats, the inclusionof a known control substance will also serve to assess assayperformance.

The economical benefit and the experimental necessity of determiningand/or quantitating two or more substances, e.g. analytes, in a testsample were the two main underlying motives in the development of amultiple-tracer assay system of the present invention. It was furtherenvisioned that an ideal multiple-tracer system would emitmultiple-wavelength signals under identical chemical conditions. It wasrecognized that it would be less desirable and more cumbersome tocombine two chemiluminescent tracers in a multiple-analyte assay systemthat required two different sets of signal generating mechanisms,conditions and timings as would be in the case of utilizing twodifferent classes of chemiluminescent compounds such as acridiniumcompounds pairing with luminol series or with stable dioxetanes, whichinvolve the use of other chemicals or enzymes to generate the signal.Furthermore the two or more different chemiluminescent compounds orconjugates must have emission efficiency differing by not more than oneorder of magnitude.

A still further fundamental requirement was the adequate stability ofthe chemiluminescent compounds in aqueous media or environment, whichwill withstand the shipping conditions for commercial products whenplaced in kit form.

Applicant has achieved these goals by developing stable chemiluminescentanalogues within the same general class that exhibit bathochromic shiftsin their emission maxima, and emit light with comparable efficiencyunder identical chemical treatments.

Chemiluminescent acridinium compounds which are shown herein to emitblue light upon treatment with hydrogen peroxide and metal hydroxidehave been well documented. U.S. Pat. Nos. 4,745,181, 4,918,192,5,110,932, U.S. Ser. No. 07/826,186 (U.S. Pat. No. 5,227,489) and U.S.Ser. No. 07/871,601 (U.S. Pat. No. 5,241,070) describe stablepolysubstituted-aryl acridinium esters; all of which are commonlyassigned and incorporated herein by reference. Such acridinium compoundsshall be referred to generally herein as "reference acridinium esters"or "acridinium esters". Such compounds as indicated include anacridinium ring system and, depending on the use of such compounds,further include an appropriate functional group(s), e.g. for attachingthe label to a substance to form conjugates for use in a test assay,including the assays of the present invention.

Batmanghelich, et al, EP-A-0 478 626 (priority GB 2233450A (Jun. 24,1989)), described the use of acridinium compounds of varying lightemission, i.e. fast and slow durations to prepare different tracerconjugates, to achieve a "substantially simultaneous" quantitation oftwo or more different analytes. This approach, however, has severalmajor drawbacks. First, one of the acridinium esters includeselectron-withdrawing substituents on the phenolic moiety in order toachieve very short duration of light emission, i.e. complete emission oremission maxima in one second. This type of compound, e.g. aortho-dihalogenated aryl acridinium ester, however, may suffer from lackof stability in aqueous environment. Second, light emission kineticsmust be carefully examined to permit accurate correction in order todistinguish the light emission contributed individually by the twotracers during the overlapping period of light emission. The describedmethod relies on the measurement of photons emitted in two separate timewindows for sequential integration of light intensity. Unless the lightemission overlap is relatively small, such correction could be apotential source for poor assay precision, particularly for detection oftwo analytes having widely different concentrations. The requirement forsmaller light emission overlap would in turn demand the availability ofa pair of chemiluminescent compounds, one having very short and theother very long duration of light emission; and would lead to compoundswhich either have stability problems or render the dual-analyte assayunpractical due to excessively prolonged signal-collection time.

Where the signal collection time is extended, an advantage of performingtwo assays in a single test sample would be lost. It is noted that inone automated analyzer using chemiluminescent detection and/orquantitation, a first test result is reported at fifteen (15) minutesand thereafter at every twenty (20) seconds during operation, see EP-A-0502 638 (priority U.S. Ser. No. 665,196 (Mar. 4, 1991), abandoned) whichis commonly assigned and incorporated herein by reference.

Batmanghelich et al also described an acridinium compound of differentlight emission spectra to prepare different tracer conjugates. Theapproach they used was to extend the electronic conjugation of theacridinium nucleus to obtain 3-(4-carboxybutadienyl)-acridinium ester(compound 2b) with bathochromic shift of about 80 nm in the emissionmaximum as compared to the parent acridinium ester (compound 2a).Extension of electronic conjugation of the acridinium nucleus does notnecessarily lead to major bathochromic shift in the emission maximumwhich is practically needed to construct a dual-analyte immunoassays anda possible reduction in emission efficiency. No teaching was made ofbenzacridinium chemiluminescent compounds or conjugates for use in suchassays.

McCapra et al, EP-A- 0 322 926 (priority U.S. Ser. Nos. 140,040 (Dec.31, 1987), abandoned and 291,843 (Dec. 29, 1988), abandoned), suggestedthe "chemiluminescent moiety" consisting of heterocyclic ring or ringsystem with ester, amide linkages attached to one of the carbon atoms onthe ring or ring system. This chemiluminescent compound was said toinclude benz a!acridinium, benz b!acridinium, and benz c!acridinium butthe synthesis and structure of these compounds or conjugates was notdescribed. Neither emission wavelength maxima, nor light emissionefficiency of these structures were predicted; nor were the use of atleast two chemiluminescent compounds or conjugates in an assay method,nor the utility of such compounds when used in assays based on theiremission spectra.

The nomenclature of benz a!acridinium and benz b!acridinium utilized inthis disclosure is based on Rule 21.5 of Definitive Rules forNomenclature of Organic chemistry, Ed. International Union of Pure andApplied Chemistry in the 1957 REPORT OF THE COMMISSION ON THENOMENCLATURE OF ORGANIC CHEMISTRY.

According to the example given on Benz a!anthracene, the compoundarising from fusing benzene ring to the peripheral sides of theacridnium nucleus (structure below) should therefore be named accordingto whether side a, b, or c of the acridinium nucleus is fused with thebenzene ring. ##STR1##

The following abbreviations are utilized in the disclosure:

1. ABAC: angular benz a!acridinium compound

2. AFAC: aromatic ring fused acridinium compound

3. EtO: ethoxy

4. DMAE: dimethyl acridinium ester

5. DIPAE: diisopropyl acridinium ester

6. LBAC: linear benz b!acridinium compound

7. LEAC: longer emission acridinium compound

8. LEAE: longer emission acridinium ester

9. MeO: methoxy

10. NSE: N-sulfoethyl

11. NSP N-sulfopropyl

12. PCT: percent cross talk

13. PMP: paramagnetic particles

14. QAE: quaternary ammonium ethoxy

15. RLU: relative light units

SUMMARY OF THE INVENTION

A method is described for detection and/or quantitation of at least twosubstances in a test sample comprising simultaneously detecting theemission signals of at least two chemiluminescent conjugates; eachchemiluminescent conjugate being associated with a substance sought tobe detected and/or quantitated in the test sample. The emission signalsof each of the chemiluminescent conjugates are discernable by theirspectral emissions, so that the substances may be detected and/orquantitated.

A chemiluminescent compound for use in the assays of the presentinvention is described in the formula: ##STR2## where W is carbon;

alternatively, C₇, W, C₉ or C₁₀ can be replaced with --N═;

or W can be omitted and C₇ connected to C₉, and C₇, C₉ or C₁₀ can bereplaced with --O--, --S--, --NH-- or --NR--;

Y is a branched or straight chained alkyl containing optionally up to 20carbon atoms, halogenated or unhalogenated, or a polysubstituted arylmoiety of the formula: ##STR3## R₁ is an alkyl, alkenyl, alkynyl oraralkyl containing optionally up to 20 heteroatoms;

R₂, R₃, R₉ and R₁₀ are identical or different groups selected fromhydrogen, substituted or unsubstituted aryl (ArR or Ar), halide, amino,hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --OR, --SR, --SSR,--C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R;

R₂ includes a single or multiple substituent at C₁₋₄ ;

R₂ can also be a fused aromatic ring with or without heteroatoms;

R₃ includes a single or multiple substituent at C₇, W, C₉ or C₁₀ ;

A-- is a counter ion including CH₃ SO₄ ⁻, FSO₃ ⁻, CF₃ SO₄ ⁻, C₄ F₉ SO₃⁻, CH₃ C₆ H₄ SO₃ ⁻ and halide;

X is a heteroatom including nitrogen, oxygen or sulfur, such that when Xis oxygen or sulfur Z is omitted, when X is nitrogen then Z is --SO₂--Y', and Y' is equal to Y, and where the substituents to Y and Y' donot have to be the same;

R₄ and R₈ are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol, amido,

R₅ and R₇ are any of R₃, R₉ and R₁₀ defined above;

R₆ ═--R₁₁ --R₁₂,

where R₁₁ is not required but optionally can be branched orstraight-chained alkyl, substituted or unsubstituted aryl or aralkylcontaining optionally up to 20 heteroatoms,

and R₁₂ is a leaving group or an electrophilic functional group attachedwith a leaving group or --Q--R--Nu, --Q--R(I)_(n) Nu, --Q--Nu, --R--Nuor --Nu, n is a number of at least 1, Nu is a nucleophilic group, Q is afunctional linkage, I is an ionic or ionizable group;

R₅ and R₆, and R₇ and R₇ are interchangeable; and

R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally upto 20 heteroatoms.

A chemiluminescent compound or conjugate is characterized in that uponchemical treatment the compound or conjugate emits a blue-green, green,yellow, orange and red-orange light having a discernable emissionspectra peak or maximum. In one embodiment, i.e. compound, the emissionmaxima is greater than or equal to 480 nm and in a preferred embodimentgreater than or equal to 515 nm.

An amplification method is described for target sequences, including oneor more nucleic acid sequences, in a test sample comprising providing atest sample suspected of containing one or more target sequences, addingan internal reference to said test sample, amplifying the targetsequences, providing at least two chemiluminescent conjugates, eachchemiluminescent conjugate being associated with target sequences andthe internal reference, and simultaneously detecting and/or quantitatingamplified target sequences and the internal reference by emissions ofthe chemiluminescent conjugates.

Accordingly, it is a primary object of the invention to provide a methodfor the simultaneous detection and/or quantitation of at least twosubstances in a test sample by use of at least two differentchemiluminescent compounds or conjugates each having discernableemission spectra.

Another object of the invention is to provide an assay method for thesimultaneous detection and/or quantitation of an analyte and an internalstandard or control in a single test medium or transfer tube.

Still another object of the invention is to increase the efficiency ofautomated analyzers by providing for the simultaneous performance of twoassays on a test sample in a single reaction medium or transfer tube.

A further object of the invention is to provide methods for synthesis ofchemiluminescent compounds and intermediate products which may be usedto synthesize such chemiluminescent compounds.

An object of the invention is to provide chemiluminescent, aromaticring-fused acridinium compounds (AFAC) that emit green or yellow light.

Another object of the invention is to provide chemiluminescent, aromaticring-fused acridinium compounds (AFAC) that emit green or yellow lightwith wavelength maxima or peaks greater than or equal to 515 nm.

Still another object of the invention is to provide a simultaneous dualchemiluminescent label assay.

A further object of the invention is to provide hydrophilic AFAC whichcarries one or more ionic and/or ionizable groups with or without,additionally, the reactive functional groups useful for forming covalentlinkage with other micro- or macromolecules or encapsulation insideliposomes.

An object of this invention is to provide AFAC conjugates formed betweenAFAC directly or indirectly with binding partners, e.g. biologicalmolecules.

Another object of this invention is to provide test assays involving theuse of acridinium ester and AFAC conjugates.

Still another object of the invention is to provide multianalyte assaysin which the determination of two or more analytes or substances orcombination thereof present in the sample as a mixture, can be carriedout simultaneously in the same reaction medium or transfer tube due tothe mutually non-interfering, or minimally overlapping but correctablelight signals produced by the same chemical treatments of two or moredifferent chemiluminescent tracers or compounds.

A further object of the present invention is to provide test kits forperforming dual chemiluminescent label test assays.

An object of the present invention is to provide test kits having two ormore chemiluminescent reagents for simultaneously assaying at least twosubstances in a test sample.

Another object of the invention is to provide intermediate compounds tobe utilized in the synthesis of labels for use in analytical assays.

Still another object of the present invention is to providechemiluminescent compounds having light emission spectra which spanabout 100-250 nm, for a spectral region with signal intensity above 5%of peak height.

These and other objects in view, as will be apparent to those skilled inthe art, the invention resides in the combination of elements set forthin the specification and covered by the claims appended hereto.

5. BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects and advantages of the invention will beappreciated more fully from the following further description thereofwith reference to the accompanying drawings wherein

FIGS. 1A-1N illustrate the structures of representative acridiniumesters, ABAC, and LBAC's of the present invention.

FIGS. 2A-2E illustrate emission spectra of acridinium esters and ABAC ofinvention as follows:

FIG. 2A is an emission spectrum of DMAE-Bz (50 μg in 0.5 ml ofacetonitrile);

FIG. 2B is an emission spectrum of 3-MeO-DMAE-Bz (50 μg in 0.5 ml ofacetonitrile) peak≈422 nm;

FIG. 2C is an emission spectrum of DIPAE-Bz (20 μg in 0.5 ml inacetonitrile) peak≈428 nm;

FIG. 2D is an emission spectrum for 3-QAE-DMAE-NHS (40 μg in 0.5 ml ofacetonitrile) peak≈426 nm; and

FIG. 2E is an emission spectrum of angular benz a!acridinium ester (2 mgin 0.2 ml of DMF) peak≈440 nm.

FIGS. 3A-3J illustrate emission spectra of LBAC of the present inventionas follows:

FIG. 3A is an emission spectrum of LEAE-Bz (50 μg in 0.5 ml ofacetonitrile) peak≈520 nm;

FIG. 3B is an emission spectrum of DIP-LEAE-Bz (50 μg in 0.5 ml ofacetonitrile) peak≈520 nm;

FIG. 3C is an emission spectrum of 3-EtO-LEAE-Bz (50 μg in 0.5 ml ofacetonitrile) peak≈508 nm;

FIG. 3D is an emission spectrum of 3-QAE-LEAE-Bz (100 μg in 0.5 ml ofacetonitrile) peak≈544 nm;

FIG. 3E is an emission spectrum of 2-MeO-LEAE-Bz (30 μg in 0.5 ml ofacetonitrile) peak≈550 nm;

FIG. 3F is an emission spectrum of LEAC-Bz (50 μg in 0.5 ml ofacetonitrile) peak≈520 nm;

FIG. 3G is an emission spectrum of 2-QAE-LEAE-NHS (70 μg in 0.5 ml ofacetonitrile) peak≈550 nm;

FIG. 3H is an emission spectrum of NSP-LEAE-Bz (15 μg in 0.5 ml ofacetonitrile) peak≈516 nm;

FIG. 3I is an emission spectrum of 2-MeO-NSE-LEAE-NHS (50 μg in 0.5 mlof acetonitrile) peak≈546 nm; and

FIG. 3J is an emission spectrum of 2-MeO-LEAE-Imidate (100 μg in 0.5 mlof acetonitrile) peak≈550 nm.

FIGS. 4A-4D illustrate emission spectra of mixed acridinium esters andLBAC of the present invention as follows:

FIG. 4A is an emission spectrum of mixed DMAE-Bz (10 μg) and LEAE-Bz (50μg in 0.5 ml of acetonitrile);

FIG. 4B is an emission spectrum of mixed DMAE-Bz (100 μg) and2-MeO-LEAE-Bz (25 μg in 0.5 ml of acetonitrile);

FIG. 4C is an emission spectrum of mixed 3-MeO-DMAE-Bz (50 μg) and2-MeO-LEAE-Bz (30 μg in 0.5 ml of acetonitrile); and

FIG. 4D is an emission spectrum of mixed 3-MeO-DMAE-Bz (5 μg) and2-QAE-LEAE-NHS (105 μg) in 0.5 ml of acetonitrile).

FIGS. 5A-5E illustrate transmittance curves of various optical filtersas follows:

FIG. 5A is a transmittance curve of a BG-38 optical filter;

FIG. 5B is a transmittance curve of a corion P70-450 optical filter;

FIG. 5C is a transmittance curve of a corion LL500 optical filter;

FIG. 5D is a transmittance curve of a corion laminated CS550 /CS600optical filter; and

FIG. 5E is a transmittance curve of a corion LL520 optical filter.

FIG. 6 illustrates the area of overlap between the transmittance curveof an optical filter (Corion LL500) and the emission spectra of DMAE-Bz.

FIG. 7 illustrates the area of overlap between the transmittance curveof an optical filter (Corion P70-450) and the emission spectra ofLEAE-Bz.

FIG. 8 illustrates FSH standard curve assays read on a dual PMTluminometer.

FIG. 9 illustrates LH standard curve assays read on a dual PMTluminometer.

6. DESCRIPTION OF PREFERRED EMBODIMENT

The AFAC of present invention comprise linear benz b!acridinium (LBAC),furanoacridinium, thiophenoacridinium, pyrroacridinium compounds andpyridoacridinium compounds. By virtue of the specific position of thearomatic ring fused to the acridinium nucleus LBAC was unexpectedlyfound to generate a chemiluminescent emission signal with much greaterbathochromic shift than the angular benz a!acridinium compounds (ABAC),and to the corresponding "reference acridinium esters", i.e. DMAE-Bz,DIPAE-Bz and 3-MeO-DMAE-Bz, see FIG. 1.

The general structure of AFAC is represented by Formula I: ##STR4##

The general structures of linear benz b!acridinium compounds and theisomeric, furano-, thiopheno-, pyrro- and pyrido-acridinium compoundsare shown by the Formulas II, III A-III C, and IV A-IV D respectively.##STR5##

Furanoacridinium compounds: V=O,

Thiophenoacridinium compounds: V=S and

Pyrroacridinium compounds: V=NH or NR. ##STR6##

One subclass of the AFAC contains reactive functional group(s), inaddition to the fundamental chemiluminescent compound with propertiesdescribed above, to enable formation, i.e., by covalent linkage, withbinding partners, and particularly with biological molecules, to produceconjugates useful as non-isotopic tracers in binding assays or as a keyintegral part of a multianalye assay system. Another subclass of theAFAC contains one or more ionic and/or ionizable groups which enhancethe solubility of the compounds in aqueous media and/or allow them to beencapsulated inside liposomes with low leakage. Such hydrophilic LBACcan also be modified to carry additional reactive functional groups toallow forming conjugates with other micro or macromolecules.

Preferred LBAC, Furanoacridinium compounds, Thiophenoacridiniumcompounds, Pyrroacridinium compounds and Pyridoacridinium compoundshaving the above-mentioned characteristics and being suitable forabove-described utilities include chemiluminescent compounds representedby the above Formulas I, II, III A-III C, and IV A-IV D respectively,where:

W is a carbon, Formula I becomes the LBAC class of chemiluminescentcompounds as represented by Formula II;

or W can be omitted and C7 connected to C9 and one of C7, C9 or C10 canbe replaced with --O--, --S--, --NH--, or --NR-- to form a 5-memberedaromatic ring fused linearly to the acridinium nucleus as shown inFormula III A-C;

or C7, W, C9, or C10 can be replaced with --N═ to form a 6-memberedpyrido ring fused linearly to the acridinium nucleus as shown in FormulaIV A-D;

R₁ is an alkyl, alkenyl, alkynyl, or aralkyl containing optionally up to20 heteroatoms, preferably nitrogen, oxygen, halogen, phosphorus orsulfur.

R₂, R₃, R₉ and R₁₀ are identical or different groups selected fromhydrogen, substituted or unsubstituted aryl (ArR or Ar), halide, amino,hydroxyl, nitro, sulfonate, --R, --CN, --CO₂ H, --SCN, --OR, --SR,--SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R.

R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, containing optionally upto 20 heteroatoms.

R₂ includes a single or multiple substituent(s) at C₁₋₄. R₃ includes asingle or multiple substituent(s) at C₇, W, C₉ or C₁₀.

R₂ can be also a fused aromatic ring with or without heteroatoms.

A-- is a counter ion including CH₃ SO₄ --, FSO₃ --, CF₃ SO₃ --, C₄ F₉SO₃ --, CH₃ C₆ H₄ SO₃ --, and halide.

X is a heteroatom including nitrogen, oxygen, or sulfur, when X isoxygen or sulfur Z is omitted, when X is nitrogen then Z is --SO₂ --Y'.

Y is a branched or straight chained alkyl containing optionally up to 20carbon atoms, halogenated or unhalogenated, or a polysubstituted arylmoiety of Formula V: ##STR7## Y' is equal to Y, and the substituents toY and Y' do not have to be the same.

R₄, and R₈ are alkyl, alkenyl, alkynyl, alkoxyl, alkylthio, amido,groups positioned to ensure better stability of AFAC in aqueous media orenvironment. The stability of AFAC rendering them suitable forcommercialization is attributed to the steric effect, electronic effect,or a combination thereof resulting from the presence of these twogroups. R₅ and R₇ are as recited for R₃, R₉, and R₁₀ in Formula I. Forconjugating AFAC to biological molecules, R₆ can be a leaving group oran electrophilic functional group attached with a leaving group, orfunctional groups which can be readily converted into such reactivegroups, directly attached or connected via a spacer to the ring. A"spacer" is defined as branched or straight-chained alkyl, substitutedor unsubstituted aryl or aralkyl, optionally containing up to 0-20heteroatoms. Examples of such functional groups include: ##STR8##--N═C═S, --N═C═O, --N₂ +U--, --N₃, --COOH, --U, or --SO₂ U, where U is ahalide.

Alternatively, R₆ can be a protected or unprotected nucleophilicfunctional group directly attached or connected via a spacer to Y. ThusR₆ ═--Q--R--Nu, --Q--R(I)n--Nu, --Q--Nu, --R--Nu, or --Nu, where R isdefined as above.

Q is a functional linkage arising from the covalent coupling between twofunctional groups each of which resides originally as substituents on Yand R or Nu, respectively. The introduction of Q in the construct of R₆represents a modular concept which allows the attachment of R--Nu,R(I)n--Nu or Nu directly to a preformed AFAC. Examples of Q include:

--C(O)--, --C(O)NH--, --NHC(O)--, --NHC(O)O--, --NH--, --O--, --S--,--NHC(O)NH--, --NHC(S)NH--, --C(═N+H₂)NH--, --SO₂ --, --SO₃ --,

(I)n is an ionic or ionizable group including but not limited toquaternary ammonium,

--COOH, --SO₃ H, --SO₄ H, --PO₃ H₂, and --PO₄ H₂, where n is a number ofat least 1.

The presence of the ionic or ionizable group(s) will enhance thehydrophilicity of AFAC and compatibility for its usage in aqueous media.The choice and positioning of such ionic or ionizable groups have theadvantage of enhancing the binding of the biological molecule/AFACconjugate to the corresponding binding partners of said biologicalmolecule.

Nu is a nucleophilic group on the compound that will facilitateconjugation of compound with biological molecules which may lacknucleophilic group for coupling, but may have electrophilic group or itsreadily converted precursor. Examples of the protected nucleophilicfunctional groups or groupings include:

t-Butyloxycarbonylamino and 3-(2-pyridinyldithio)propionyl (PDP).##STR9## t-Butyloxycarbonyl (t-Boc) is the protective group on aminowhich can be removed by acid, e.g. trifluoroacetic acid, treatment. TheS-2-pyridinyl group in PDP is a protective moiety which can be removedto generate free --SH group upon treatment with dithiothreotol (DTT) atsuitable pH. The usage of these protective groups of --NH₂ and --SHnucleophilic groups is known to those skilled in the arts of organicchemistry. Examples of the unprotected nucleophilic functional groupsinclude: amino, thiol, hydroxyl, active methylene adjacent to strongelectron-withdrawing group, organic metallic moieties. Examples of sucha nucleophilic R₆ grouping, its conjugation to biological molecules, andthe conjugate utilities have been disclosed in EP-A-0 361 817 (priorityU.S. Ser. No. 249,620 (Sep. 26, 1988), abandoned) which is commonlyassigned and incorporated herein by reference.

To provide more hydrophilic compounds that can be encapsulated insideliposomes for the purpose of constructing signal-enhancing lumisomes,R₂, R₃ or R₆ can be strongly ionizable groups directly attached or moresuitably connected via spacer to the aromatic rings. Examples ofstrongly ionizable groups include: phosphate, phosphonate, sulfate, andsulfonate. Examples of such a R₆ grouping, the incorporation of thehydrophilic chemiluminescent molecules into liposomes and the utility ofthe resulting lumisomes have been disclosed in EP-A-0 361 817 (priorityU.S. Ser. No. 226,639 (Aug. 1, 1988), abandoned) which is commonlyassigned and incorporated herein by reference. Similarly, to providehydrophilic AFAC that can be conjugated with biological moleculesdirectly, R₂ and/or R₃ can be ionic or strongly ionizable groupsdirectly attached or more suitably connected via spacer to thearomatic-ring fused acridinium nucleus, and R₆ can be reactivefunctional group-containing side chain as recited above. The positionsof R₂, R₃, R₅ and R₆, and R₆ and R₇ substituents in all AFAC areinterchangeable.

One of the possible precursors to AFAC should be AFAC with the R₆substituent being hydrogen or R, with R defined as above.

When X is nitrogen, Y can be a branched or straight chained alkyl of 1to 20 carbon atoms or a moiety equal to Formula V above with all thepossible substituents as recited, and Z is represented by the followingFormula VI:

    Z═--SO2--Y'                                            (Formula VI)

where Y' is a branched or straight chained alkyl of 1 to 20 carbonatoms, halogenated or unhalogenated, or a moiety equal to the Formula Vshown above with all the possible substituents as recited. Thesubstituents to both Y and Y' do not necessarily have to be the same.

A preferred aromatic ring-fused acridinium compounds (AFAC) should be asdescribed above. More preferentially, they are the LBAC series with thefollowing substituents: R₁ is a methyl, sulfopropyl or sulfoethyl group;R₉, R₁₀ are hydrogen, methoxy or halogen; R₂ is a hydrogen, 2-MeO,2-quarternaryammoniumalkoxy, 3-MeO, 3-EtO, 3-quarternaryammoniumalkoxy,or 3-carboxyalkyloxy group; R₃, R₅ and R₇ are hydrogen; when X is oxygenor sulfur, R₄ and R₈ are methyl, ethyl, isopropyl groups; R₆ is one ofthe following groups attached to the 4-position of Formula V,carboxylate, N-succinimidyloxycarbonyl, benzyloxycarbonyl,N-aminoalkylcarbamoyl, Sulfomethylcarbamoyl, N-N-(2-amino-3-S-(3'-sulfopropyl)-thiopropionyl)-2-aminoethyl!carbamoyl,N-7-(1,3-disulfonaphthalenyl)carbamoyl, N-1-carboxyl-2-(3-sulfopropylthio)ethyl!carbamoyl,N-(2-sulfonyloxyethyl)carbamoyl, N-(2-phosphonoethyl)carbamoyl,N-(2-phosphonoxyethyl)carbamoyl and alkoxyiminoethyl; when X isnitrogen, Y represents Formula V with R₅ and R₇ being hydrogen and R₆being carboxylate, N-succinimidyloxycarbonyl,N-succinimidyloxycarbonylalkyl, benzyloxycarbonyl, orN-aminoalkylcarbamoyl; Z represents Formula VI with Y' being an alkyl orphenyl.

Intermediate compounds which may be utilized to synthesize thechemiluminescent compounds of the present invention include:

An intermediate of the formula: ##STR10## where w is carbon;alternatively, C₇, W, C₉ or C₁₀ can be replaced with --N═;

or W can be omitted and C₇ connected to C₉, and C₇, C₉ or C₁₀ can bereplaced with --O--, --S--, --NH-- or --NR--;

R₂, R₃, R₉ and R₁₀ are identical or different groups selected fromhydrogen, substituted or unsubstituted aryl (ArR or Ar), halide, amino,hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --R, --OR, --SR,--SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R;

R₂ includes a single or multiple substituent at C₁₋₄ ;

R₃ includes a single or multiple substituent at C₇, W, C₉ and C₁₀ ;

R₂ can also be a fused aromatic ring with or without heteroatoms; and

R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally upto 20 heteroatoms.

An intermediate of the formula: ##STR11## where W is carbon;alternatively, C₇, W, C₉ or C₁₀ can be replaced with --N═;

or W can be omitted and C₇ connected to C₉, and C₇, C₉ or C₁₀ can bereplaced with --O--, --S--, --NH-- or --NR--;

Y is a branched or straight chained alkyl containing optionally up to 20carbon atoms, halogenated or unhalogenated, or a polysubstituted arylmoiety of the formula: ##STR12## R₂, R₃, R₉ and R₁₀ are identical ordifferent groups selected from hydrogen, substituted or unsubstitutedaryl (ArR or Ar), halide, amino, hydroxyl, nitro, sulforate, --R, --CN,--COOH, --SCN, --OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or--NHC(O)R;

R₂ includes a single or multiple substituent at C₁₋₄ ;

R₃ includes a single or multiple substituent at C₇, W, C₉ or C₁₀ ;

R₂ can also be a fused aromatic ring with or without heteroatoms;

X is a heteroatom including nitrogen, oxygen or sulfur, such that when Xis oxygen or sulfur Z is omitted, when X is nitrogen then Z is --SO₂--Y', Y' is equal to Y and the substituents to Y and Y' do not have tobe the same;

R₄ and R₈ are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol, amido;

R₅ and R₇ are any of R₃, R₉ and R₁₀ defined above;

R₆ ═--R₁₁ --R₁₂,

where R₁₁ is not required but optionally can be branched orstraight-chained alkyl, substituted or unsubstituted aryl or aralkylcontaining optionally up to 20 heteroatoms;

and R₁₂ is a leaving group or an electrophilic functional group attachedwith a leaving group or --Q--R--Nu, --Q--R(I)_(n) Nu, --Q--Nu, --R--Nuor Nu where n is a number of at least 1, Nu is a nucleophilic group, Qis a functional linkage, I is an ionic or ionizable group;

R₅ and R₆, and R₆ and R₇ are interchangeable; and

R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally upto 20 heteroatoms.

The following examples describe the synthesis of the preferred compoundsand intermediates of the present invention, the structures of which areshown in FIG. 1. The examples are intended to illustrate and not tolimit the invention and may be used as a guide by those skilled in theart to synthesize compounds having alternate substituents than thoseshown in the examples.

EXAMPLE 1 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl5-Methyl-benz b!acridinium-12-carboxylate Fluorosulfonate (LEAE-Bz)

3-Anilino-2-naphthoic Acid

A mixture of 3-hydroxy-2-naphthoic acid (Aldrich cat. #H4600-7) (376 g,2.0 mol) and aniline (376 ml, 4.1 mol) was heated at 170° C. withstirring under nitrogen for 16 hours. The resulting mixture, when hot,was poured into 1N HCl (2.5 ml), heated to 100° C., and stirred at thistemperature for 5 minutes. The mixture, when hot, was filtrated and thesolid was washed with 0.2N HCl (600 ml). The wet material was boiled andmechanically stirred with 0.5N sodium carbonate solution (6.0 ml) for 10minutes, cooled and filtered. The reddish filtrate was treated dropwisewith 5N HCl with stirring to .sup.˜ pH 7. The resulting yellowprecipitate was collected, washed with small amount of water andcrystallized from ethanol (400 ml) to give 3-anilino-2-naphthoic acid(36 g, 7%). Rf 0.6 (silica gel, EM Art. 5715, 20% methanol/chloroform).MS (EI): m/z 264 (M).

12-Chloro-benz b!acridine

A mixture of 3-anilino-2-naphthoic acid (10.0 g, 37.98 mmol) andphosphorous oxychloride (35.4 ml, 379.8 mmol) was refluxed at 150° C.under nitrogen with stirring for 2 hours. The resulting purple mixturewas cooled and evaporated under reduced pressure to dryness. The contentwas added with stirring to a mixture of chloroform/ice/conc. ammoniumhydroxide (200 ml/200 g/200 ml). The chloroform layer was separated anddried over calcium chloride. Removal of the solvent under reducedpressure gave 12-chlorobenz b!acridine (9.4 g, 95%). Rf 0.8 (silica gel,hexane/ethyl acetate 2:1). MS (EI): m/z 263 (M).

12-Cyano-benz b!acridine

A mixture of 12-chloro-benz b!acridine (2.3 g, 8.68 mmol), potassiumcyanide (620 mg, 9.55 mmol) and copper(I) cyanide (391 mg, 4.43 mmol) inanhydrous methanol (16 ml) was bubbled with nitrogen for 1 minute andthen kept in a sealed tube. The mixture was heated at 160° C. withstirring for 4.5 hours and cooled. The red-brown mixture was evaporatedand the residue was flash-chromatographed (W. C. still et al: J. Org.Chem., 43, 2923, (1978)) on a silica column (Baker silica gel, Cat#7024-1) packed with hexane and eluted with 10% ethyl acetate-hexane,yielding red 12-cyano-benz b!acridine (1.54 g, 70%). Rf 0.7 (silica gel,hexane/ethyl acetate 2:1). MS (FAB, Thioglycerol Matrix): m/z 255 (M+1).

Benz b!acridine-12-carboxylic Acid Hydrochloride

A mixture of 12-cyano-benz b!acridine (557 mg, 2.19 mmol) andtetrabutylammonium bromide (71 mg, 0.22 mmol) in 50% sulfuric acid (v/v,50 ml) was heated at 160°-170° C. under nitrogen with stirring for 44hours and cooled. The resulting mixture was poured into ice-water (500ml); the purple precipitate was collected and washed with water. The wetmaterial was dissolved with warming in 2N NaOH (100 ml) and thenfiltered. The filtrate was acidified in an ice-water bath withconcentrated HCl to pH 3-4, giving purple benz b!acridine-12-carboxylicacid hydrochloride (510 mg, 75%). Rf 0.4 (silica gel,chloroform/methanol/water 65:25:4). MS (FAB, Thioglycerol Matrix): m/z274 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl Benz b!acridine-12-carboxylate

A suspension of benz b!acridine-12-carboxylic acid hydrochloride (370mg, 1.2 mmol) in anhydrous pyridine (50 ml) was warmed at 60° C. for 5minutes. The slightly cloudy solution was then cooled to 0° C. andtreated with p-toluenesulfonyl chloride (388 mg, 2.33 mmol) at 0° C. for10 minutes and at room temperature for another 15 minutes to give thefirst reaction mixture. This reaction mixture was further treated withbenzyl 2,6-dimethyl-4-hydroxybenzoate, see U.S. Pat. No. 4,745,181, (694mg, 2.71 mmol) to give the second reaction mixture, which was stirred atroom temperature under nitrogen for 40 hours, and then evaporated underreduced pressure to dryness. The residue was flash-chromatographed on asilica column packed with hexane and eluted with 50% ether-hexane togive orange-red (4-benzyloxycarbonyl-2,6-dimethyl)phenyl benzb!acridine-12-carboxylate (450 mg), 74%). Rf 0.6 (silica gel, 20% ethylacetate/toluene). MS (FAB, Thioglycerol Matrix): m/z 512 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 5-Methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (LEAE-Bz)

To a solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl benzb!acridine-9-carboxylate (115 mg, 0.23 mmol) in anhydrous methylenechloride (5 ml) was added methyl fluorosulfonate (0.128 ml, 2.25 mmol).The solution was stirred at room temperature under nitrogen for 20hours, and then treated with anhydrous ether (10 ml). The resultingprecipitate was collected and washed with ether (100 ml), givingdark-brown (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (136 mg, 97%). MS (FAB,Thioglycerol Matrix): m/z 526 (M).

EXAMPLE 2 Preparation of(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 5-Methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (LEAE-NHS)

(4-Carboxy-2,6-dimethyl)phenyl Benz b!acridine-12-carboxylateHydrobromide

A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl benzb!acridine-12-carboxylate (198 mg, 0.39 mmol), prepared in Example 1, in30% hydrogen bromide-acetic acid (5 ml) was stirred at 55°-60° C. undernitrogen for 4 hours. The mixture was treated with anhydrous ether (20ml); the precipitate was collected and washed with ether (100 ml) togive (4-carboxy-2,6-dimethyl)phenyl benz b!acridine-12-carboxylatehydrobromide quantitatively. Rf 0.4 (silica gel, 5%methanol/chloroform). MS (EI): m/z 421 (M).

(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl Benzb!acridine-12-carboxylate

To a solution of (4-carboxy-2,6-dimethyl)phenyl benzb!acridine-12-carboxylate hydrobromide (108 mg, 0.22 mmol) in anhydrousN,N-dimethylformamide (5 ml) was added at 0° C. dicyclohexylcarbodiimide(111 mg, 0.54 mmol). After stirring at this temperature for 30 minutes,N-hydroxysuccinimide (62 mg, 0.54 mmol) was added. The solution wasstirred under nitrogen at 0° C. for 10 minutes and then at roomtemperature for 24 hours. The resulting mixture was treated with aceticacid (3 drops) and evaporated to dryness under reduced pressure. Theresidue was extracted with chloroform and the chloroform extract wasevaporated under reduced pressure to dryness. The residue wasflash-chromatographed on a silica column packed and eluted with ether togive (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl benzb!acridine-12-carboxylate (20 mg, 18%). Rf 0.6 (silica gel, 20%). MS(EI): m/z 518 (M).

(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 5-Methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (LEAE-NHS)

To a solution of (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl benzb!acridine-12-carboxylate (14 mg, 0.026 mmol) in anhydrous methylenechloride (1 ml) was added methyl fluorosulfonate (0.021 ml, 0.26 mmol).The resulting brown solution was stirred at room temperature undernitrogen for 20 hours, and then treated with anhydrous ether (1 ml). Theprecipitate was collected and washed with ether (40 ml), yielding(2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate-fluorosulfonate (11 mg, 65%). MS (FAB,Thioglycerol Matrix): m/z 533 (M)

EXAMPLE 3 Preparation of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl5-Methyl-benz b!acridinium-12-carboxylate Fluorosulfonate (DIP-LEAE-Bz)

3,5-Diisopropyl-4-hydroxybenzoic acid

This acid was prepared according to the procedure of W. H. Meek et al.J. Chemical and Engineering Data, 14(3), 388, (1969). To a solution of2,6-diisopropylphenol (Aldrich cat. #D12660-8) (37.0 ml, 0.20 mol) inanhydrous N,N-dimethylacetamide (150 ml) was added sodium methoxide(16.2 g, 0.30 mol). Carbon dioxide was passed through the mixturethroughout the subsequent reaction period. The mixture was heated withstirring and the solvent was slowly distilled out during 2 hours of theperiod until the pot temperature reached to 180° C. The mixture wasallowed to continue stirring at 180° C. for another 1.5 hours and thencooled to 90° C. The flow of carbon dioxide was discontinued and water(400 ml) was added. The mixture, after further cooled to roomtemperature, was washed with toluene (4×60 ml) and then treated withconc. hydrochloric acid in an ice-water bath to pH 3. Theresulting-mixture was extracted with diethyl ether (2×150 ml); the etherextract was washed with brine (100 ml) and dried over anhydrousmagnesium sulfate. Removal of the solvent under reduced pressure gave3,5-diisopropyl-4-hydroxybenzoic acid (10.4 g, 23%). Rf 0.5 (silica gel,50% diethyl ether/hexane). MS (CI, CH4): m/z 223 (M+1).

Benzyl 3,5-Diisopropyl-4-hydroxybenzoate

To a solution of 3,5-diisopropyl-4-hydroxybenzoic acid (1.223 g, 5.50mmol) in methanol (25 ml) was added potassium hydroxide (308 mg, 5.50mmol) in water (5 ml). The resulting solution was stirred at roomtemperature under nitrogen for 1 hour, and then evaporated completely todryness under reduced pressure. This potassium salt was dissolved inanhydrous acetonitrile (30 ml) and N,N-dimethylformamide (15 ml), andtreated with dibenzo-18-crown-6 (198 mg, 0.55 mmol). After 30 minutes ofstirring at 80° C. under nitrogen, the solution was further treated withbenzyl bromide (0.712 ml, 6.05 mmol). The stirring was continued at 80°C. under nitrogen for 4 hours. The resulting mixture, after cooling, wasfiltrated. The filtrate was evaporated under reduced pressure todryness. The residue was flash-chromatographed on a silica column packedwith hexane and eluted with 20% ethyl acetate/hexane, yieldingcrystalline benzyl, 3,5-diisopropyl-4-hydroxybenzoate (1.35 g, 79%). Rf0.6 (silica gel, 20% ethyl acetate/toluene). MS(EI). m/z 312 (M).

(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl Benzb!acridine-12-carboxylate

A suspension of benz b!acridine-12-carboxylic acid hydrochloride fromExample 1 (200 mg, 0.65 mmol) in anhydrous pyridine (30 ml) was warmedat 60° C. for 5 minutes. The slightly cloudy solution was then cooled to0° C. and treated with p-toluenesulfonyl chloride (247 mg, 1.29 mmol).The solution was stirred at 0° C. for 40 minutes and room temperaturefor another 10 minutes, and benzyl 2,6-diisopropyl-4-hydroxybenzoate(202 mg, 0.65 mmol) was added. This reaction mixture was allowed tocontinue stirring at room temperature under nitrogen for 20 hours, andthen evaporated under reduced pressure to dryness. The residue wasflash-chromatographed on a silica column packed with hexane and elutedwith 25% ether-hexane to give orange(4-benzyloxycarbonyl-2,6-diisopropyl)phenyl benzb!acridine-12-carboxylate (187 mg, 51%). Rf 0.6 (silica gel, 20% ethylacetate/toluene). MS(CI, CH4): m/z 570 (M+3).

(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 5-Methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (DIP-LEAE-Bz)

To a solution of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl benzb!acridine-12-carboxylate (50 mg, 0.088 mmol) in anhydrous methylenechloride (3 ml) was added methyl fluorosulfonate (0.072 ml, 0.088 mmol).The brown solution was stirred at room temperature under nitrogen for 24hours, and then treated with anhydrous ether (4 ml). The resultingprecipitate was collected and washed with ether (10 ml), givingdark-brown (4-benzyloxycarbonyl-2,6-diisopropyl)phenyl 5-Methyl-benzb!acridinium-12-carboxylate fluorosulfonate (39 mg, 64%). MS (FAB,Thioglycerol Matrix): m/z 582 (M).

EXAMPLE 4 Preparation of N-(4-Methoxyphenyl-N-3-(benzyloxycarbonyl)phenylsulfonyl!5-Methyl-benzb!acridinium-12-carboxamide Fluorosulfonate (LEAC-Bz)

3- N-(4-Methoxyphenyl)sulfamido!benzoic acid

To a solution of 3-(chlorosulfonyl)benzoic acid (Kodak cat. #1188655)(4.4 g, 20.00 mmol) and triethylamine (2.78 ml, 20.00 mmol) in anhydrousmethylene chloride (40 ml) was added at 0° C. 4-anisidine (2.46 g, 20.00mmol). After 10 minutes of stirring at 0° C., a large quantity ofprecipitate formed from the solution. The mixture was allowed tocontinue stirring at room temperature under nitrogen for 2 hours. Afterfiltration, the collected off-white solid was washed with water (50 ml)and then with ether (50 ml), giving 3-N-(4-methoxyphenyl)sulfamido!benzoic acid (4.50 g, 74%). Rf 0.5 (silicagel, chloroform/methanol/water 65:25:4). MS(CI, CH4): m/z 308 (M+1).

Benzyl 3- N-(4-Methoxyphenyl)sulfamido!benzoate

To a solution of 3- N-(4-methoxyphenyl)sulfamido!benzoic acid (2.00 g,6.52 mmol) in N,N-dimethylformamide (20 ml) was added a solution ofsodium hydroxide (260.6 mg, 6.52 mmol) in water (5 ml). The resultingsolution was stirred at room temperature under nitrogen for 1 hour, andthen evaporated completely to dryness under reduced pressure. Thissodium salt was dissolved in anhydrous acetonitrile (40 ml) andN,N-dimethylformamide (20 mL), and treated with dibenzo-18-crown-6 (235mg, 0.65 mmol). After 30 minutes of stirring at 80° C. under nitrogen,the solution was further treated with benzyl bromide (0.852 ml, 7.27mmol). The stirring was continued at 80° C. under nitrogen for 4 hours.The resulting mixture, after cooling, was filtrated. The white solid waswashed with small amount of acetonitrile. The combined acetonitrilefiltrate was evaporated under reduced pressure to dryness. The residewas flash-chromatographed on a silica column packed with hexane andeluted with 15% ethyl acetate/hexane, yielding crystalline benzyl 3-N-(4-methoxyphenyl)sulfamido!benzoate (2.00 g, 77%). Rf 0.5 (silica gel,20% ethyl acetate/toluene). MS(CI, CH4): m/z 397 (M+1).

N-(4-Methoxyphenyl)-N- 3-(benzyloxycarbonyl)phenylsulfonyl!Benzb!acridine-12-carboxamide

A suspension of benz b!acridine-12-carboxylic acid hydrochloride (200mg, 0.65 mmol) in anhydrous pyridine (30 ml) was warmed at 60° C. for 5minutes. The slightly cloudy solution was then cooled to 0° C. andtreated with p-toluenesulfonyl chloride (247 mg, 1.29 mmol). Thesolution was stirred at 0° C. for 40 minutes and room temperature foranother 10 minutes, and benzyl 3- N-(4-methoxyphenyl)sulfamido!benzoate(257 mg, 0.65 mmol) was added. This reaction mixture was allowed tocontinue stirring at room temperature under nitrogen for 20 hours, andthen was evaporated under reduced pressure to dryness. The residue wasflash-chromatographed on a silica column packed with hexane and elutedwith 25% hexane-ether to give orange N-(4-methoxyphenyl)-N-3-(benzyloxycarbonyl)phenylsulfonyl!benz b!acridine-12-carboxamide (177mg, 68%). Rf 0.4 (silica gel, 20% ethyl acetate/toluene). MS(CI, CH4):m/z 653 (M+1).

N-(4-Methoxyphenyl)-N- 3-(benzyloxycarbonyl)phenylsulfonyl!5-Methyl-benzb!acridinium-12-carboxamide Fluorosulfonate (LEAC-Bz)

To a solution of N-(4-methoxyphenyl)-N-3-(benzyloxycarbonyl)phenylsulfonyl!benz b!acridine-12-carboxamide (50mg, 0.077 mmol) in anhydrous methylene chloride (3 ml) was added methylfluorosulfonate (0.062 ml, 0.77 mmol). The dark-brown solution wasstirred at room temperature under nitrogen for 20 hours, and treatedwith anhydrous ether (10 ml). The resulting precipitate was collectedand washed with ether (10 ml), giving blue-black N-(4-methoxyphenyl)-N-3-(benzyloxycarbonyl)phenylsulfonyl!5-methyl-benzb!acridinium-12-carboxamide fluorosulfonate (38 mg, 65%). MS (FAB,Thioglycerol Matrix): m/z 667 (M).

EXAMPLE 5 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-Ethoxy-5-methyl-benz b!acridinium-12-carboxylate Fluorosulfonate(3-EtO-LEAE-Bz)

Benzo 5,6!isatin

Benzo 5,6!isatin was prepared according to the procedure of A. Etienneand A. Staehelin, Bull. Soc. Chim. France., 6, 743, (1954).

3-Hydroxy-benz b!acridine-12-carboxylic Acid.

A mixture of benzo 5,6!isatin (500 mg, 2.54 mmol) and potassiumhydroxide (996 mg, 17.78 mmol) in water (2 ml) and n-butanol (2 ml) washeated with stirring to 100° C. to give a homogeneous solution, followedby addition of resorcinol (1.95 g, 17.78 mmol). After the solution wasfurther heated to 140° C. and the solvents were slowly blown away withnitrogen during a 30-minute period, another 2 ml of water and 1 ml ofn-butanol were added. The temperature of the solution was maintained at140° C. while blowing of nitrogen continued for a total of 2 hours. Thegummy mixture was cooled and dissolved in 100 ml of water; the solutionwas acidified with concentrated hydrochloric acid in an ice-water bathto pH 2. The resulting precipitate was collected, washed with water, andflash-chromatographed on a silica column packed with chloroform andeluted with 20% methanol-chloroform followed bychloroform-methanol-water (65:25:4) to give 3-hydroxy-benzb!acridine-12-carboxylic acid (210 mg, 29%). Rf 0.3 (silica gel,chloroform/methanol/water 65:25:4). MS (FAB, Thioglycerol Matrix): m/z290 (M+1).

Ethyl 3-Ethoxy-benz b!acridine-12-carboxylate

To a mixture of 3-hydroxy-benz b!acridine-12-carboxylic acid (90 mg,0.28 mmol) and cesium carbonate (451 mg, 1.38 mmol) in methyl sulfoxide(3.5 ml) was added bromoethane (207 ul, 2.77 mmol). The mixture wasstirred under nitrogen at 25° C. for 4 hours, and treated with water (10ml). The mixture was adjusted to pH 5 with 5% HCl; the resultingprecipitate was collected and washed with water. The crude product waspurified on a silica column packed with chloroform and eluted with 5%methanol-chloroform to give ethyl 3-ethoxy-benzb!acridinium-12-carboxylate (36 mg, 38%). Rf 0.5 (silica gel, diethylether/hexane 3:1). MS (FAB, Thioglycerol Matrix): m/z 346 (M+1).

3-Ethoxy-benz b!acridinium-12-carboxylic Acid Hydrochloride

A solution of ethyl 3-ethoxy-benz b!acridinium-12-carboxylate (35 mg,0.10 mmol) in 16% sodium hydroxide (1 ml) and methanol (3 ml) wasstirred under nitrogen at 25° C. for 2 days and at 40° C. for additional4 hours, and then evaporated to dryness under reduced pressure. Theresidue was suspended in water (20 ml) and adjusted to pH 3 in anice-water bath with concentrated HCl. The precipitate was collected andwashed with water to give 3-ethoxy-benz b!acridine-12-carboxylic acidhydrochloride (30 mg, 83%). Rf 0.8 (silica gel,chloroform/methanol/water 65:25:4). MS (CI, CH4): m/z 318 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 3-Ethoxy-benzb!acridine-12-carboxylate

A slightly-cloudy solution of 3-ethoxy-benz b!acridine-12-carboxylicacid hydrochloride (19 mg, 0.054 mmol) in pyridine (1 ml) andN,N'-dimethylpropyleneurea (DMPU, 1.5 ml) was cooled at 0° C. andp-toluenesulfonyl chloride (21 mg, 0.11 mmol) was added. After 20minutes of stirring, benzyl 3,5-dimethyl-4-hydroxybenzoate (14 mg, 0.055mmol) and N,N'-dimethylaminopyridine (1 mg) were added. The mixture wasstirred at 25° C. under nitrogen for 20 hours and then evaporated underreduced pressure to dryness. The residue was treated with water (5 ml)and extracted with ether (5×5 ml). The combined ether layer was washedwith water (1×10 ml), brine (1×10 ml) and dried over magnesium sulfate.Removal of the solvent under reduced pressure gave a crude mixture,which was separated on a preparative-TLC plate (2 mm silica gel, EM Art.5717) by developing with ether/hexane (3:2). The major orange band wascollected and extracted with 10% methanol/ether. Removal of the solventsunder reduced pressure gave orange(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 3-ethoxy-benzb!acridine-12-carboxylate (5 mg, 17%). Rf 0.6 (silica gel, diethylether/hexane). MS (FAB, Thioglycerol Matrix): m/z 556 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 3-Ethoxy-5-methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (3-EtO-LEAE-Bz)

To a solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 3-ethoxy-benzb!acridine-12-carboxylate (9 mg, 0.0162 mmol) in methylene chloride (1ml) was added methyl fluorosulfonate (13.1, 0.162 mmol). The solutionwas stirred at 25° C. under nitrogen for 36 hours and then treated withdiethyl ether (10 ml). The precipitate was collected and washed withdiethyl ether (20 ml), giving(4-benzyloxycarbonyl-2,6-dimethyl)phenyl-3-ethoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (3-EtO-LEAE-Bz, 7 mg, 65%)MS (CI, CH4): m/z 570 (M+2).

EXAMPLE 6 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-(N,N-Diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridnium-12-carboxylate Difluorosulfonate (3-QAE-LEAE-Bz )

N,N-Diethylaminoethyl 3-(N,N-Diethylamino)ethoxy-benzb!acridine-12-carboxylate

A solution of 3-hydroxy-benz b!acridine-12-carboxylic acid (415 mg, 1.28mmol) in methyl sulfoxide (12 ml) was treated with cesium carbonate (5g, 15.4 mmol) at 25° C. for 10 minutes and diethylamminoethyl bromidehydrobromide (1.5 g, 6.4 mmol) was added. The mixture was stirred at 25°C. under nitrogen for 4 hours and then quenched with water (100 ml). Theprecipitate was collected and washed with water (50 ml). The crudeproduct was separated on 4 preparative-TLC plates (2 mm silica gel) bydeveloping with 20% methanol/chloroform. The orange band was collectedand extracted with 10% methanol/chloroform. Removal of the solventsunder reduced pressure gave N,N-diethylaminoethyl3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylate (73 mg, 12%).Rf 0.6 (silica gel, 20% methanol/chloroform). MS (CI, CH4): m/z 488(M+1).

3-(N,N-Diethylamino)ethoxy-benz b!acridine-12-carboxylic acidhydrochloride

A solution of N,N-diethylaminoethyl 3-(N,N-diethylamino)ethoxy-benzb!acridine-12-carboxylate (60 mg, 0.123 mmol) in 4N sodium hydroxide (4ml) and methanol (12 ml) was stirred at 65° C. under nitrogen for 16hours, and then evaporated under reduced pressure to dryness. Theresidue was dissolved in water (10 ml); the solution was carefullyacidified in an ice-water bath with concentrated HCl to pH 4. Theresulting precipitate was collected and washed with diethyl ether (5 ml)to give 3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylic acidhydrochloride (32 mg, 61%). Rf 0.3 (silica gel,chloroform/methanol/water 65:25:4).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 3-(N,N-Diethylamino)ethoxy-benzb!acridine-12-carboxylate

A mixture of 3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylicacid hydrochloride (29 mg, 0.069 mmol) and p-toluenesulfonyl chloride(29 mg, 0.152 mmol) in pyridine (12 ml) was stirred at 80° C. for 5minutes. The resulting homogeneous solution was cooled to 25° C. andfurther treated with benzyl 4-hydroxy-3,5-dimethylbenzoate (20 mg, 0.078mmol). After stirring at 25° C. under nitrogen for 16 hours, the solventwas removed under reduced pressure. The residue was purified on apreparative-TLC plate (2 mm silica gel) developed with 15%methanol/chloroform. The orange band was collected and extracted with10% methanol/chloroform. Evaporation of the solvents under reducedpressure gave (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylate (17 mg, 39%).Rf 0.6 (silica gel, 10% methanol/chloroform). MS (FAB, Glycerol Matrix):m/z 627 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-(N,N-Diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridinium-12-carboxylate Difluorosulfonate (3-QAE-LEAE-Bz)

A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylate (13 mg, 0.0208mmol) in methylene chloride (1.9 ml) was treated with methylfluorosulfonate (25 ul, 0.308 mmol). After 15 hours of stirring undernitrogen at 25° C., the reaction mixture was slowly added to diethylether (5 ml). The precipitate was collected and washed with ether (10ml) to give (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-(N,N-diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridinium-12-carboxylate difluorosulfonate (3-QAE-LEAE-Bz, 9 mg,53%). MS (FAB, Glycerol Matrix): m/z 659 (M+3).

EXAMPLE 7 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl2-Methoxy-5-methyl-benz b!acridinium-12-carboxylate Fluorosulfonate(2-MeO-LEAE-Bz)

3-(4-Methoxy) anilino-2-naphthoic Acid

A mixture of p-anisidine (84.7 g, 687.7 mmol) and 3-hydroxy-2-naphthoicacid (64.7 g, 343.9 mmol) was mechanically stirred at 160° C. undernitrogen for 22 hours. After cooling to 130° C., the mixture was treatedwith hot 1N hydrochloric acid (1000 ml), stirred at 130° C. for 10minutes, and filtrated, when hot. The resulting cake was stirred withhot 0.5N sodium carbonate (2200 ml) for 15 minutes and filtrated whenhot. The filtrate was cooled and acidified to pH 6.5 with concentratedHCl in an ice-water bath. The precipitate was collected and washed withmethanol (150 ml), yielding 3-(4-methoxy)anilino-2-naphthoic acid (14.8g, 15%). Rf 0.6 (silica gel, 10% methanol/chloroform). MS (CI, CH4): m/z294 (M+1).

12-Chloro-2-methoxy-benz b!acridine

A mixture of 3-(4-methoxy)anilino-2-naphthoic acid (15.4 g, 49.44 mmol)and phosphorousoxy chloride (46 ml, 494.4 mmol) was refluxed at 120° C.under nitrogen for 3.5 hours, and then evaporated under reduced pressureto dryness. The residue was taken into a mixed solvent containingchloroform (500 ml)/ice (450 g)/ammonium hydroxide (450 ml). Theresulting two layers were separated. The aqueous layer was extractedwith chloroform (3×250 ml). The combined chloroform layer was dried overcalcium chloride and evaporated to dryness under reduced pressure,yielding 12-chloro-2-methoxy-benz b!acridine (12.5 g, 86%). Rf 0.8(silica gel, 60% diethyl ether/hexane). MS (CI, CH4): m/z 294 (M+1).

12-Cyano-2-methoxy-benz b!acridine

A mixture of 12-chloro-2-methoxy-benz b!acridine (562 mg, 1.905 mmol),potassium cyanide (136 mg, 2.096 mmol) and copper(I) cyanide (86 mg,0.953 mmol) in methanol. (3.7 ml) was stirred at 170° C. in asealed-tube for 4.5 hours. The resulting mixture was filtrated, and thesolid was washed with chloroform/methanol (2:1, 10 ml). The combinedfiltrate was evaporated under reduced pressure to give a residue, whichwas flash-chromatographed on a silica column packed with chloroform andeluted with 1% methanol/chloroform to yield 12-cyano-2-methoxy-benzb!acridine (477 mg, 88%). Rf 0.6 (silica gel, 1% methanol/chloroform).MS (CI, CH4): m/z 285 (M+1).

2-Hydroxy-benz b!acridine-12-carboxylic acid hydrosulfate

A mixture of 12-cyano-2-methoxy-benz b!acridine (8.3 g, 29.1 mmol) and50% sulfuric acid (v/v, 280 ml) was mechanically stirred under nitrogenat 160° C. for 48 hours. The resulting mixture was cooled and pouredinto ice-water (1800 ml). The precipitate was collected, washed withwater (200 ml), and flash-chromatographed on a silica column packed withchloroform and eluted with 20% methanol/chloroform followed bychloroform/methanol/water (65:25:4) to give 2-hydroxy-benzb!acridine-12-carboxylic acid hydrosulfate (4.8 g, 43%). Rf 0.4 (silicagel, chloroform/methanol/water 65:25:4).

Methyl 2-Methoxy-benz b!acridine-12-carboxylate

To a solution of 2-hydroxy-benz b!acridine-12-carboxylic acidhydrochloride (186 mg, 0.572 mmol) in methyl sulfoxide (4 ml) were addedcesium carbonate (746 mg, 2.29 mmol) and iodomethane (143 ul, 2.29mmol). The resulting mixture was stirred at 25° C. under nitrogen for 4hours and then treated with water (50 ml). The mixture was acidified inan ice-water bath with concentrated HCl to pH 6. The resultingprecipitate was collected, washed with water (5 ml) and air-dried. Thecrude mixture was purified on 4 preparative-TLC plates (2 mm silica gel)developed with diethyl ether/hexane (5:1); the major orange band wascollected and extracted with 10% methanol/chloroform. Removal of thesolvents under reduced pressure gave methyl 2-methoxy-benzb!acridine-12-carboxylate (35 mg, 17%). Rf 0.8 (silica gel, diethylether/hexane 5:1).

2-Methoxy-benz b!acridine-12-carboxylic Acid

A solution of methyl 2-methoxy-benz b!acridine-12-carboxylate (35 mg,0.10 mmol) in 4N sodium hydroxide (3 ml) and methanol (9 ml) was stirredat 65° C. for 15 hours. The resulting mixture was evaporated underreduced pressure to dryness. The residue was dissolved in water (40 ml);the aqueous solution was acidified to pH 5 with concentrated HCl in anice-water bath. The precipitate was collected and washed with water (5ml), yielding 2-methoxy-benz b!acridine-12-carboxylic acid (20 mg, 59%).Rf 0.5 (silica gel, chloroform/methanol/water 65:25:4). MS (CI, CH4: m/z304 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 2-Methoxy-benzb!acridine-12-carboxylate

A solution of 2-methoxy-benz b!acridine-12-carboxylic acid (18 mg,0.0529 mmol) in pyridine (5 ml) was treated with p-toluenesulfonylchloride (20 mg, 0.106 mmol) at 25° C. for 15 minutes and then benzyl3,5-dimethyl-4-hydroxy-benzoate (27 mg, 0.106 mmol) was added. After 15hours of stirring at 25° C. under nitrogen, the reaction mixture wasevaporated under reduced pressure to remove the pyridine. The residuewas purified on a preparative-TLC (2 mm silica gel) developed withdiethyl ether/hexane (3:2). The orange band was collected and extractedwith 5% methanol/chloroform. Removal of the solvents under reducedpressure gave (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 2-methoxy-benzb!acridine-12-carboxylate (2.7 mg, 9%). Rf 0.5 (silica gel, 60% diethylether/hexane).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 2-Methoxy-5-methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (2-MeO-LEAE-Bz)

A solution of 4-benzyloxycarbonyl-2,6-dimethyl)phenyl 2-methoxy-benzb!acridine-12-carboxylate (2.5 mg, 0.0046 mmol) in methylene chloride (1ml) was treated with methyl fluorosulfonate (3.7 ul, 0.56 mmol) at 25°C. with stirring under nitrogen for 15 hours. The reaction mixture wasadded to anhydrous diethyl ether (4 ml). The resulting precipitate wascollected and washed with diethyl ether (5 ml) to afford(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (2-MeO-LEAE-Bz) (1.5 mg,49%). MS (FAB, Thioglycerol Matrix): m/z 556(M).

EXAMPLE 8 Preparation of (2,6-Dimethyl-4-succinimidyloxycarbonyl)phenyl5-Methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylateDifluorosulfonate (2-QAE-LEAE-NHS)

N,N-Dimethylaminoethyl 2-(N,N-Dimethylamino)ethoxy-benzb!acridine-12-carboxylate

To a solution of 2-hydroxy-benz b!acridine-12-carboxylic acidhydrochloride (360 mg, 1.108 mmol) in methyl sulfoxide (11 ml) wereadded cesium carbonate (3.61 g, 11.08 mmol) and N,N-dimethylaminoethylbromide hydrobromide (1.03 g, 4.432 mmol). After 15 hours of stirring at60° C. under nitrogen, the reaction mixture was diluted with methylsulfoxide (20 ml) and filtered to remove the insoluble impurities. Thefiltrate was concentrated under reduced pressure to a small volume,which was separated on a preparative-TLC plate (2 mm silica gel) bydeveloping with chloroform/methanol/water (47:48:5). The desired orangeband was collected and extracted with 25% methanol/chloroform. Removalof the solvents under reduced pressure gave N,N-dimethylaminoethyl2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylate (86 mg, 18%).Rf 0.6 (silica gel, chloroform/methanol/water 65:25:4). MS (FAB,Glycerol Matrix): m/z 432 (M+1).

2-(N,N-Dimethylamino)ethoxy-benz b!acridine-12-carboxylic acid

A solution of N,N-dimethylaminoethyl 2-(N,N-dimethylamino)ethoxy-benzb!acridine-12-carboxylate (86 mg, 0.20 mmol) in 4N sodium hydroxide (7.3ml) and methanol (22 ml) was stirred at 65° C. for 1 hour and at 35° C.for 15 hours. The reaction mixture was evaporated under reduced pressureto dryness. The residue was washed with water (5 ml) and air-dried,yielding 2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylic acid(23 mg, 32%). Rf 0.3 (silica gel, chloroform/methanol/water 65:25:4). MS(FAB, Glycerol Matrix): m/z 361 (M+1).

Succinimidyl 3,5-Dimethyl-4-hydroxy-benzoate

A solution of 3,5-dimethyl-4-hydroxybenzoic acid (5.0 g, 30.0 mmol) inN,N-dimethylformamide (150 ml) was cooled to 0° C. and treated withN-hydroxysuccinimide (3.45 g, 30.0 mmol) and1,3-dicyclohexylcarbodiimide (6.81 g, 33.0 mmol). The solution wasstirred under nitrogen at 0° C. for 2 hours and then at 25° C. for 16hours. The resulting mixture was stirred with 0.5 ml of acetic acid for15 minutes, and then filtered to remove the insoluble urea. The filtratewas evaporated under reduced pressure to dryness. The dried material waswashed with diethyl ether (100 ml) and suspended in boiling ethylacetate (200 ml). The suspension, when hot, was filtered to removeinsoluble impurities. The filtrate was concentrated and suspended in hotethyl acetate/methylene chloride (1:1, 200 ml) and cooled to give anoff-white powder in 2.91 g (37%). Rf 0.6 (silica gel, diethyl ether).

(2,6-Dimethyl-4-succinimidyloxycarbonyl)phenyl2-(N,N-Dimethylamino)ethoxy-benz b!acridine-12-carboxylate

A solution of 2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylicacid (30 mg, 0.0833 mmol) in pyridine (4 ml) was treated withp-toluenesulfonyl chloride (31.8 mg, 0.166 mmol) at 25° C. for 10minutes, followed by addition of succinimidyl3,5-dimethyl-4-hydroxybenzoate (32 mg, 0.0833 mmol). After 15 hours ofstirring at 25° C. under nitrogen, the solution was diluted withchloroform (10 ml), quickly washed with water (3×4 ml) and evaporatedunder reduced pressure to dryness. The residue was purified on twopreparative-TLC plates (1 mm silica gel) developed with 10%methanol/chloroform. The desired orange band was collected and extractedwith 10% methanol/chloroform. Removal of the solvents under reducedpressure gave (2,6-dimethyl-4-succinimidyloxycarbonyl)phenyl2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylate (7 mg, 14%).Rf 0.8 (silica gel, 10% methanol/chloroform). MS (FAB, ThioglycerolMatrix): m/z 606 (M+1).

(2,6-Dimethyl-4-succinimidyloxycarbonyl)phenyl5-Methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylateDifluorosulfonate (2-QAE-LEAE-NHS)

A solution of 2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl2-(N,N-dimethyl amino)ethoxy-benz b!acridine carboxylate (1.9 mg, 0.0031mmol) in anhydrous methylene chloride (4 ml) was treated with methylfluorosulfonate (3.8 ul, 0.0465 mmol). The solution was allowed to stirat 25° C. under nitrogen for 16 hours. The resulting precipitate wascollected and washed with diethyl ether (2 ml) to give(2,6-dimethyl-4-succinimidyloxycarbonyl)phenyl5-methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylatedifluorosulfonate (2-QAE-LEAE-NHS) (1.0 mg, 39%).

EXAMPLE 9 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl5-(3-Sulfopropyl)-benz b!acridinium-12-carboxylate (NSP-LEAE-Bz)

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 5-(3-Sulfopropyl)-benzb!acridinium-12-carboxylate (NSP-LEAE-Bz)

A mixture of (4-benzylcarboxyl-2,6-dimethyl)phenyl benzb!acridine-12-carboxylate from Example 1 (30 mg, 0.0587 mmol) and1,3-propane sulton (600 mg, 4.9 mmol) was flushed with nitrogen and keptin a sealed tube. The tube was heated with stirring at 180° C. for 5hours and then cooled. The resulting mixture was purified byreverse-phase preparative-HPLC on a C-18 column (YMC SH-344-15, S-15,128), eluted under gradient condition with 25% to 40% acetonitrile in0.05M aqueous trifluoroacetic acid from 0 to 20 minutes, 40% to 90% over5 minutes, and maintaining 90% acetonitrile for another 10 minutes. Thefraction with retention time of 17 minutes was collected and evaporatedunder reduced pressure to give 3.2 mg of the title compound(NSP-LEAE-Bz). MS (FAB, Glycerol Matrix): m/z 634 (M+1).

EXAMPLE 10 Preparation of(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl2-Methoxy-5-(2-sulfoethyl)-benz b!acridinium-12-carboxylate(2-MeO-NSE-LEAE-NHS)

(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 2-Methoxy-benzb!acridinium-12-carboxylate

A mixture of 2-methoxy-benz b!acridine-12-carboxylic acid from Example 7(208 mg, 0.6118 mmol) in pyridine (20 ml) was treated withp-toluenesulfonyl chloride (233 mg, 1.2235 mmol) at 25° C. for 10minutes, followed by addition of succinimidyl3,5-dimethyl-4-hydroxy-benzoate (161 mg, 0.6118 mmol). After 24 hours ofstirring under nitrogen at 25° C., the pyridine was removed byevaporation under reduced pressure. The resulting mixture wasflash-chromatographed on a silica column packed and eluted with diethylether. The crude product collected was further purified on aChromatotron plate (1 mm silica gel) by elution with diethyl ether toyield 80 mg (24%) of the pure product. Rf 0.8 (silica gel, ethylacetate/hexane 2:1). MS (FAB, Thioglycerol Matrix): 549 (M+1).

2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl2-Methoxy-5-(2-sulfoethyl)-benz b!acridinium-12-carboxylate(2-MeO-NSE-LEAE-NHS)

A mixture of (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl2-methoxy-benz b!acridine-12-carboxylate (23 mg, 0.04197 mmol) andethylenesulfonyl chloride (prepared according to the procedure of C. S.Rondestredt Jr., J. Amer. Chem. Soc., 76, 1926 (1954)) (378 ul, 4.197mmol) was stirred at 25° C. under nitrogen for 63 hours. The resultingmixture was purified by reverse-phase preparative-HPLC on a C-18 column,eluted under gradient condition with 35% acetonitrile in 0.05M aqueoustrifluoroacetic acid from 0 to 10 minutes, 35% to 80% from 10 to 30minutes, 80% to 90% from 30 to 35 minutes, and remaining 90% for another5 minutes. The fraction with retention time of 25 minutes was collectedand evaporated under reduced pressure to give 2.2 mg (8%) of the titlecompound (2-MeO-NSE-LEAE-NHS). MS (FAB, Thioglycerol Matrix): m/z 657(M+1).

EXAMPLE 11 Preparation of 2,6-Dimethyl-4-(2-methoxyiminoethyl)!phenyl2-Methoxy-5-methyl-benz b!acridinium-12-carboxylate Dichloride(2-MeO-LEAE-Imidate)

(4-Cyanoethyl-2,6-dimethyl)phenyl 2-Methoxy-benzb!acridine-12-carboxylate

A suspension of 2-methoxy-benz b!acridine-12-carboxylic acid fromExample 7 (100 mg, 0.2941 mmol) in pyridine (15 ml) was treated withp-toluenesulfonyl chloride (112 mg, 0.5882 mmol) at 0° C. for 40minutes, followed by addition of triethylamine (164 ul. 1.1789 mmol) and4-cyanoethyl-2,6-dimethyl-phenol (prepared according to the procedure ofE. Jexova et al, CS 158810, Jul. 15, 1975; CA 84(13):898299)(51 mg,0.2914 mmol). After 18 hours of stirring at 25° C. under nitrogen, thereaction solution was evaporated under reduced pressure to remove thepyridine. The residue was purified on 4 preparative-TLC plates (2 mmsilica gel) developed twice with ethyl acetate/hexane (3:4). The majororange band was collected and extracted with methanol/chloroform (1:30).Removal of the solvents under reduced pressure gave the title compound(75 mg, 55%). Rf 0.5 (silica gel, ethyl acetate/hexane 2:3). MS (FAB,Thioglycerol Matrix): m/z 461 (M+1).

(4-Cyanoethyl-2,6-dimethyl)phenyl 2-Methoxy-5-methyl benzb!acridinium-12-carboxylate Fluorosulfonate

A solution of (4-cyanoethyl-2,6-dimethyl)phenyl 2-methoxy-benzb!acridine-12-carboxylate (20 mg, 0.04338 mmol) in methylene chloride (1ml) was treated with methyl fluorosulfonate (17.5 ul, 0.2169 mmol) at25° C. with stirring under nitrogen for 18 hours. The reaction mixturewas added to anhydrous diethyl ether (5 ml). The resulting precipitatewas collected and purified by reverse-phase preparative-HPLC on a C-18column, eluted under gradient condition with 40% to 80% acetonitrile in0.05M aqueous trifluoroacetic acid from 0 to 30 minutes, and remaining80% acetonitrile for another 40 minutes. The fraction with retentiontime of 24 minutes was collected and evaporated under reduced pressureto give the product (15.7 mg, 65%). MS (FAB, Thioglycerol Matrix): m/z475 (M).

2,6-Dimethyl-4-(2-methoxyiminoethyl)!phenyl 2-methoxy-5-methyl benzb!acridinium-12-carboxylate Dichloride (2-MeO-LEAE-Imidate)

A solution of (4-cyanoethyl-2,6-dimethyl)phenyl 2-Methoxy-5-methyl benzb!acridinium-12-carboxylate fluorosulfonate (4 mg, 0.00697 mmol) inanhydrous methanol (0.5 ml) was treated with anhydrous hydrogen chloride(gas) at 0° C. for 10 minutes. The reaction solution was then reduced byblowing with nitrogen to a small volume; and the concentrate was addedto anhydrous diethyl ether (3 ml). The resulting precipitate wascollected and washed with diethyl ether (5 ml), yielding the titlecompound (2-MeO-LEAE-Imidate) (1.5 mg, 37%).

PREPARATION OF THE ANALOGS OF ACRIDINIUM ESTER

EXAMPLE 12 Preparation of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl10-Methyl-acridinium-9-carboxylate Fluorosulfonate (DIPAE-Bz)

(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl Acridine-9-carboxylate

A mixture of acridine-9-carboxylic acid hydrochloride (74 mg, 0.33 mmol)in thionyl chloride (3 ml, 41.1 mmol) was refluxed at 110° C. undernitrogen for 2 hours. After cooling, the solution was evaporated underreduced pressure to dryness. The solid was washed with anhydrous diethylether (5 ml) to give acridine-9-carbonyl chloride hydrochloride. Thisacid chloride was dissolved in anhydrous pyridine (4 ml), followed byaddition of benzyl 3,5-diisopropyl-4-hydroxy-benzoate (102 mg, 0.33mmol) and 4-N,N-dimethylamino-pyridine (16 mg, 0.13 mmol). After 16hours of stirring at 25° C. under nitrogen, the solution was evaporatedunder reduced pressure to dryness. The residue was purified on aChromatotron plate (1 mm silica gel) by elution with 20% diethylether/hexane to yield (4-benzyloxycarbonyl-2,6-diisopropyl)phenylacridine-9-carboxylate (64 mg, 38%). Rf 0.6 (silica gel, 20% ethylacetate/toluene). MS(EI): m/z 517(M).

(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl10-Methyl-acridinium-9-carboxylate Fluorosulfonate (DIPAE-Bz)

A solution of (4-benzyloxycarbonyl-2,6-diisopropyl)phenylacridine-9-carboxylate (62 mg, 0.120 mmol) in anhydrous methylenechloride (3 ml) was treated with methyl fluorosulfonate (97 ul, 1.198mmol). After 21 hours of stirring at 25° C. under nitrogen, the solutionwas treated with anhydrous diethyl ether (10 ml). The resultingprecipitate was collected, washed with diethyl ether (20 ml) andcrystallized from acetonitrile/diethyl ether to give(4-benzyloxycarbonyl-2,6-diisopropyl)phenyl10-methyl-acridinium-9-carboxylate fluorosulfonate (DIPAE-Bz) (20 mg,26%)

EXAMPLE 13 Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-Methoxy-10-methyl-acridinium-9-carboxylate Fluorosulfonate(3-MeO-DMAE-Bz)

Methyl 3-Methoxy-acridine-9-carboxylate

To a solution of 3-hydroxy-acridine-9-carboxylic acid (2 g, 8.368 mmol)in methyl sulfoxide (50 ml) was added at 25° C. cesium carbonate (10.9g, 33.47 mmol), followed by slow addition of iodomethane (2.08 ml, 33.47mmol). After 2 hours of stirring at 25° C. under nitrogen, the mixturewas poured into water (500 ml). The precipitate was collected, washedwith water (200 ml) and air-dried. The resulting mixture wasflash-chromatographed on silica column packed with chloroform and elutedwith 1% methanol/chloroform, followed by 2% methanol/chloroform, to givethe crude product. This crude product was further purified on sixpreparative-TLC plates (2 mm silica gel) by elution with 5%methanol/chloroform. The major band was collected and extracted with 5%methanol/chloroform. Removal of the solvents under reduced pressure gavemethyl 3-methoxy-acridine-9-carboxylate (1.05 g, 47%). Rf 0.7 (silicagel, 5% methanol/chloroform).

3-Methoxy-acridine-9-carboxylic Acid Hydrochloride

A solution of methyl 3-methoxy-acridine-9-carboxylate (900 mg, 3.37mmol) in 4N sodium hydroxide (10 ml) and methanol (30 ml) was stirred at65° C. under nitrogen for 14 hours, cooled and evaporated under reducedpressure to dryness. The solid was dissolved in water (100 ml); theaqueous solution was washed with diethyl ether (4×50 ml) and acidifiedin an ice-water bath with concentrated HCl to pH 3. The resultingprecipitate was collected, washed with water (200 ml) and air-dried, togive 3-methoxy-acridine-9-carboxylic acid hydrochloride (710 mg, 73%).Rf 0.6 (silica gel, chloroform/methanol/water 65:25:4).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-Methoxy-acridine-9-carboxylate

To a suspension of 3-methoxy-acridine-9-carboxylic acid hydrochloride(150 mg, 0.519 mmol) in pyridine (25 ml) was added at 0° C.p-toluenesulfonyl chloride (198 mg, 1.038 mmol). After stirred for 10minutes, the suspension turned homogeneous; and then benzyl3,5-dimethyl-4-hydroxybenzoate (132 mg, 0.519 mmol) was added. Thesolution was stirred at 65° C. under nitrogen for 2 hours and at 25° C.for additional 20 hours, and evaporated under reduced pressure todryness. The residue was suspended in chloroform (100 ml), washed with5% ammonium hydroxide (4×50 ml), water (2×50 ml), brine (1×50 ml) anddried over anhydrous magnesium sulfate. Removal of the chloroform underreduced pressure gave a crude mixture, which was purified on 2preparative-TLC plates (2 mm silica gel) developed with toluene/ethylacetate (4:1). The major band was collected and extracted with 10%methanol/chloroform. Evaporation of the solvents under reduced pressureyielded (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-methoxy-acridine-9-carboxylate. Rf 0.7 (silica gel, 20% ethylacetate/toluene). MS: (CI CH₄) m/z 492 (M+1).

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl3-Methoxy-10-methyl-acridinium-9-carboxylate Fluorosulfonate(3-MeO-DMAE-Bz)

A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-methoxy-acridine-9-carboxylate (45 mg, 0.0916 mmol) in anhydrousmethylene chloride (2 ml) was treated with fluoromethyl sulfonate (74ul, 0.916 mmol). After 19 hours of stirring at 25° C. under nitrogen,the solution was treated with anhydrous diethyl ether (6 ml). Theresulting precipitate was collected and washed with diethyl ether (20ml), yielding (4-benzyloxycarbonyl-2,6-dimethyl)phenyl3-methoxy-10-methyl-acridinium-9-carboxylate fluorosulfonate(3-MeO-DMAE-Bz) (41 mg, 74%). MS (FAB, Thioglycerol Matrix): m/z 506(M).

EXAMPLE 14 SYNTHESIS OF AN ABAC

The preparations of an angular benz a!acridinium ester,(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 5-methyl-benza!acridinium-12-carboxylate methosulfate and the intermediates are givenbelow:

Benz a!acridine-12-carboxylic acid

The procedure is essentially that reported by Martinet, J. and Dansette,A. in Bull. Soc. Chim., Fr., 45, 101 (1929).

N-Phenyl-β-naphthylamine (22.9 g, 0.1 mol) (Aldrich, Cat #17,805-5) wasmixed with diethylketomalonate (18 g, 0.1 mol) (Aldrich, Cat #D9,740-1)in 5 ml of acetic acid and heated in an oil bath at 150° C. for 45 mins.The reaction mixture solidified upon cooling. The solid was transferredto a fritted funnel, washed thoroughly with ethyl alcohol, and dried ina desiccator under vacuum to give ethylphenyl-1-benzo-4,5-dioxindol-3-carboxylate: mp 169°-170° C. A portion ofthis first intermediate (7 g, 0.02 mol) was further treated with 100 mlof 10% KOH, heated at reflux for 90 minutes and left at room temperatureovernight. To this second reaction mixture was added 200 ml of 1N HCl.The resulting yellow precipitate was filtered, washed with boilingethanol, dried to give 2.1 g (33.8%) of the title compound. MS (CI,CH4): m/z 274 (M+1).

Benz a!acridine-12-carbonyl chloride

To benz a!acridine-12-carboxylic acid (1 g, 3.66 mmol) obtained abovewas added 10 ml of thionyl chloride. The mixture was heated at 95° C.for 3 hours, cooled, treated with 50 ml of benzene, and stored at 4° C.overnight. The precipitates were collected by filtration, washed withbenzene, then ethyl ether, and dried in a desiccator under vacuum togive 300 mg (28%) of the acid chloride.

(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 5-methyl-benza!acridinium-12-carboxylate methosulfate

A solution of 4-benzyloxycarbonyl-2,6-dimethylphenol (0.27 g, 1 mmol) in10 ml of dry pyridine was treated with 32 mg, 0.26 mmol of4-dimethylaminopyridine (Aldrich, Cat.#10,770-0). To this solution wasadded benz a!acridine-12-carbonyl chloride prepared above. The solutionwas heated at 100° C. for 3 hours and evaporated to give a residue whichwas purified on 3 preparative TLC plates (EM Cat #5717) developed with5% methanol in toluene/ethyl acetate (4:1) mixture. A fluorescent bandwith Rf slightly below that of the starting phenol was stripped, elutedwith 5% methanol in chloroform, and the eluent evaporated to give 433 mgof yellow intermediate, (4-benzyloxycarbonyl-2,6-dimethyl)phenyl benza!acridine-12-carboxylate.

This intermediate was dissolved in 20 ml of trichloromethane, treatedwith 4 ml of dimethyl sulfate and heated at 85° C. for 48 hours andcooled. The yellow precipitate was filtered and washed with ether togive 222 mg (37%) of the desired product. MS (FAB Thioglycerol Matrix):m/z 526 (M)).

PREPARATION OF CONJUGATES

In the chemiluminescent compounds of the present invention, preferablyat the R₆ position, depending on which coupling moiety is selected, theAFAC label can be reacted directly with the specific binding partner,ligand, or hapten either in an aqueous or an organic medium.

It is understood that alternate positions of the chemiluminescentcompound may have a coupling moiety to be reacted with a binding partnerto form a conjugate.

The chemiluminescent labels can include an appropriate leaving group oran electrophilic functional group attached with a leaving group orfunctional groups which can be readily converted into such reactivegroups, directly attached or connected via a spacer for attaching asubstance to form a conjugate to be utilized in a test assay. An exampleof preparing the LEAE-anti-TSH conjugate is provided below.

Preparation of LEAE-Anti-TSH conjugate

A solution of a monoclonal anti-TSH antibody (2 mg, 0.013 umol) in 1.36ml of 0.1M phosphate buffer, pH 8.0 was treated with a solution ofLEAE-NHS (43 ug, 0.067 umole) in 240 ul of acetonitrile at roomtemperature for one hour. The conjugation reaction was stopped by addinga solution of lysine (10 mg) in 0.5 ml of 0.1M phosphate buffer, pH 8.The LEAE-conjugated anti-TSH was purified by passing the reactionmixture through a Sephadex G-25 column (1×20 cm) packed and eluted with10 mM Phosphate, pH 8. The elution was monitored at 280 nm with a ISCOUV detector. The desired conjugate was collected when the first voidvolume peak was eluted out.

Preparation of Oligonucleotide conjugate

A method for conjugating binding parties, haptens, or ligands ofluminescent labels to polynucleotides is described in EP-A-0 537 994(priority U.S. Ser. No. 775,399, filed Oct. 16, 1991, abandoned), whichis commonly assigned and incorporated herein by reference.

LIGHT EMISSION SPECTRA

The light emission spectra of LBAC's and the reference acridinium esterswere determined by a Fast Spectral Scanning System (FSSS) of PhotoResearch (a division of Kollmorgen Corp) of Burbank, Calif., U.S.A. Theexperiment was carried out in a dark room. Each sample was dissolved inHPLC grade acetonitrile at the concentration of 1 mg/ml or higher anddiluted with the same solvent to obtain the sample solution in theconcentration specified. A typical determination utilized 10 to 100 ugof each compound, with the exception of the angular benz a!acridiniumester (2 mg), separately or mixed together in 0.5 ml acetonitrilecontained in 13×100 mm borosilicate test tube. The tube was placed on atube rack raised to a proper height. The FSSS optical head was placed infront of the tube at close distance and with its lense focused on theliquid in the tube. The sample solution was first treated with 0.35 mlof the Flashing Reagent #1 (Ciba Corning Diagnostics) containing 0.1NHNO₃ and 0.1% H₂ O₂. The room was then darkened, and 0.35 ml of theFlashing Reagent #2 (Ciba Corning Diagnostics) containing 0.25N NaOH and0.2% ARQUAD was added to the reaction mixture immediately, see U.S. Pat.No. 4,927,769 which is commonly assigned and incorporated herein byreference. The light which was generated instantaneously following theaddition of the Reagent #2 was recorded by FSSS for 4 seconds except for2-MeO-LEAE-Imidate which was recorded for 30 seconds starting from splitsecond before the Reagent #2 was added. The results of the variousdeterminations are summarized in Table I.

                  TABLE I                                                         ______________________________________                                                               Emission Max                                                                              Range*                                     Compound      Quantity .sup.˜ (nm)                                                                         (nm)                                       ______________________________________                                        1.  DMAE-Bz       20     ug  426-428   410-510                                2.  3-MeO-DMAE-Bz 50     ug  422       395-520                                3.  DIPAE-Bz      20     ug  426       405-520                                4.  ABAC          2      mg  436-440   410-530                                5.  LEAE-Bz       50     ug  520-524   490-670                                6.  DIP-LEAE-Bz   50     ug  520       485-670                                7.  2-MeO-LEAE-Bz 30     ug  550       510-700                                8.  3-EtO-LEAE-Bz 50     ug  508       470-660                                9.  3-QAE-LEAE-Bz 100    ug  544       470-680                                10. 2-QAE-LEAE-NHS                                                                              70     ug  550       510-700                                11. LEAC-Bz       50     ug  520       485-670                                12. NSP-LEAE-Bz   15     ug  516       482-655                                13. 2-MeO-NSE-    50     ug  546       500-700                                    LEAE-NHS                                                                  14. 2-MeO-LEAE-   100    ug  550       500-710                                    Imidate                                                                   ______________________________________                                         .sup.˜ The emission maximum for each compound could vary by 0-4 nm      between different determinations.                                             *Range is set for spectral region with signal intensity of above 5% of        peak height.                                                                   The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl                          5methyl-benz a!acridinium12-carboxylate methosulfate.                    

Recorded emission spectra are shown in FIGS. 2A-2E, 3A-3J, and 4A-4D.FIGS. 2A-2E and 3A-3J show individual emission spectra ofchemiluminiscent compounds including an acridinium ring system andcompounds including a benzacridinium ring system. The difference of theemission maxima between acridinium esters and LBAC's were found to rangebetween 80-128 nm, while that between acridinium esters and the ABAC wasabout 8-14 nm. As shown in FIGS. 4A-4D, when the acridinium esters andLBAC's were mixed in a tube and flashed simultaneously, the resultingcombined emission spectra showed the ideal summed up spectral profile,indicative of the non-interfering nature of these two groups ofchemiluminescent emission signals. It is understood that these data mayvary depending on the instrumentation utilized and the components of theinstrumentation, particularly the filters. The major portions of theoriginal constituting spectra which remained unchanged were indeednon-overlapping. These important physical characteristics fulfill theprerequisite for two or more subclasses of chemiluminescent compounds tobe utilized in test assays for detecting and/or quantitating at leasttwo substances in a test sample, and particularly to multianalyteclinical diagnostic assays. In the preferred method a benzacridiniumcompound is utilized as one component of the assay method and morespecifically an N-alkylated benzacridinium compound.

As noted above, a luminometer for detecting and/or quantitating at leasttwo chemiluminescent emission spectra is described in U.S. Ser. No.08/035,341, abandoned.

LIGHT EMITTING EFFICIENCY

The light emitting efficiency of LBAC's, ABAC, and DMAE-Bz wasdetermined on a Berthold luminometer (MLA-I) (Ciba Corning DiagnosticsCorp.) fitted with a BG-38 filter with wavelength transmission range ofabout 320 to 650 nm at transmission efficiency of 20 to 97%. (FIG. 5,Panel A). Alternate filters may be incorporated in luminometers toexpand the range of transmission efficiency.

Each sample was prepared in acetonitrile solution at 1 mg/ml, seriallydiluted to 10 ug/ml in acetonitrile and further on to 1 ng/ml, 0.1 ng/mland 0.01 ng/ml in 10 mM phosphate buffer iwht 0.15M NaCl, 0.1% BSA.0.05% NAN₃, pH8.

To determine the light emitting efficiency, 25 ul of blank (the buffermatrix) or each sample were flashed by injecting 0.35 ml each of theFlashing Reagent #1 and #2 sequentially. Light emission was integratedfor 2 seconds and results as means of duplicate determination are givenin Table II.

                  TABLE II                                                        ______________________________________                                                                             RLU's/                                   Compound   Mole-   Total Counts (RLU's)/2 sec                                                                      mol*                                     (counter   cular   amount flashed (pg)                                                                             (1 ×                               ion)       Weight  0.25 pg 2.5 pg 25.0 pg                                                                              E20)                                 ______________________________________                                        DMAE-Bz    587     76,477  769,477                                                                              6,786,57                                                                             1.8                                  (CH.sub.3 SO.sub.4.sup.-)                                                     DIPAE-Bz   631     82,115  845,660                                                                              6,041,380                                                                            2.1                                  (FSO.sub.3.sup.-)                                                             3-MeO-DMAE-Bz                                                                            605     --      54,760 523,380                                                                              0.13                                 (FSO.sub.3.sup.-)                                                             ABAC       613     --      23,600 143,400                                                                              0.058                                (CH.sub.3 SO.sub.4.sup.-)                                                     LEAE-Bz    625     105,857 1,037,947                                                                            9,037,063                                                                            2.6                                  (FSO.sub.3.sup.-)                                                             DIP-LEAE-Bz                                                                              681     29,930  240,610                                                                              2,413,320                                                                            0.66                                 (FSO.sub.3.sup.-)                                                             LEAC-Bz    766     79,873  767,553                                                                              6,312,163                                                                            2.4                                  (FSO.sub.3.sup.-)                                                             3-EtO-LEAE-Bz                                                                            669     93,635  883,785                                                                              6,364,935                                                                            2.4                                  (FSO.sub.3.sup.-)                                                             3-QAE-LEAE-Bz                                                                            854     16,765  107,540                                                                              1,005,855                                                                            0.37                                 (FSO.sub.3.sup.-)                                                             2-MeO-LEAE-Bz                                                                            655     27,810  223,995                                                                              2,140,890                                                                            0.59                                 (FSO.sub.3.sup.-)                                                             2-QAE-LEAE-NHS                                                                           854     ND.sup.˜                                                                        ND.sup.˜                                                                       ND.sup.˜                                                                       ND.sup.˜                       (FSO.sub.3.sup.-)                                                             NSP-LEAE-Bz                                                                              633     22,715  212,485                                                                              2,250,520                                                                            0.54                                 2-MeO-NSE- 646     14,853  145,403                                                                              1,503,100                                                                            0.38                                 LEAE-NHS                                                                      2-MeO-LEAE-                                                                              578     ND.sup.˜                                                                        ND.sup.˜                                                                       ND.sup.˜                                                                       ND.sup.˜                       Imidate (2 Cl.sup.˜)                                                    ______________________________________                                         *counts/mol calculated from quantity of 2.5 pg.                                The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl                          5methyl-benz a!acridinium12-carboxylate methosulfate.                         .sup.˜ ND = not determined prior to the establishment of final          purity of the compound.                                                  

From the data shown in Table II, the light emitting efficiency of theLBAC's was comparable to that of DMAE-Bz within the range of 0.21 to1.39 fold, depending on the substituents on the benzacridinium nucleusand the phenoxy group. It should be noted these determinations werebased on 2-second signal collection and have not taken into account theflashing kinetics of the individual compounds, e.g. some compounds maytake greater than 2 seconds to release most of their signals, thesensitivity of the photomultiplying tube, and the transmissionefficiency of the optical filter(s) at different points of the spectralrange. These findings, however, were totally unexpected in view of themuch lower light emitting efficiency of the isomeric ABAC. This level oflight emitting efficiency renders LEAC's useful in sensitive bindingassays, including multi-analyte assays.

KINETIC STUDY ON LIGHT EMISSION

Due to the electronic and/or steric effects of different substituents onthe phenoxy moiety, the acridinium and benzacridinium nucleus, it wasanticipated that not all the DMAE analogs, ABAC and LEAC's would havethe same flashing rates under identical conditions. In other words,within 2 seconds of signal collection time different compounds wereexpected to release different percentages of total releasable signals. Atime course study over a period of up to 10 seconds was conducted todetermine these percentages, by flashing the compounds and normalizingall the signals collected for different lengths of time to that of 10seconds. The results are summarized in Table III.

                  TABLE III                                                       ______________________________________                                                  Percent signal released over                                                  different lengths of time                                           Compounds   10.0 s  6.0 s   4.0 s                                                                              2.0 s 1.0 s                                                                              0.5 s                             ______________________________________                                        DMAE-Bz     100%    99%     96%  80%   48%  10%                               DIPAE-Bz    100%    98%     97%  89%   64%  14%                               3-MeO-DMAE-Bz                                                                             100%    80%     70%  57%   49%  26%                               ABAC        100%    89%     73%  52%   20%  3%                                LEAE-Bz     100%    98%     97%  88%   71%  27%                               DIP-LEAE-Bz 100%    80%     67%  47%   26%   4%                               LEAC-Bz     100%    102%    98%  93%   82%  73%                               3-EtO-LEAE-Bz                                                                             100%    95%     93%  85%   78%  45%                               3-QAE-LEAE-Bz                                                                             100%    91%     88%  84%   75%  34%                               2-MeO-LEAE-Bz                                                                             100%    92%     83%  65%   42%  16%                               2-QAE-LEAE-NHS                                                                            100%    88%     76%  59%   37%   9%                               NSP-LEAE-Bz 100%    95%     96%  91%   72%  14%                               2-MeO-NSE-  100%    96%     90%  79%   58%  22%                               LEAE-NHS                                                                      2-MeO-LEAE- 100%    76%     59%  40%   23%   6%                               Imidate                                                                       ______________________________________                                          The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl                          5methyl-benz a!acridinium12-carboxylate methosulfate.                    

As shown by the data of TABLE III, particularly at the 0.5 and 1 secondintervals, the flashing kinetics varied widely for different DMAEanalogs, ABAC and LEAC's. These data on release percentages should beutilized in comparing the light emission efficiency of the compounds fordeveloping various assay utilizing the chemiluminescent compounds.

MUTUALLY NON-INTERFERING LIGHT EMISSION

Beside exhibiting discernable mutually non-interfering nature of theirlight emission spectra as mentioned above, DMAE and LEAE in the form ofprotein conjugates also demonstrated no mutual interactions in theirlight emissions during flashing as shown by no decrease or increase ofthe combined Relative Light Units (RLU) registered.

The testing was carried out as follows:

DMAE-anti-TSH and LEAE-anti-TSH were diluted in 10 mM phosphate bufferwith 0.15M NaCl, 0.1% BSA, 0.05% NaN₃, pH 8 at two concentrations, suchthat 25 ul of the solutions would give about ₂₀₀,000 and 1,000,000RLU's, respectively, when they were flashed in the same manner on theBerthold luminometer equipped as described above. The light emission ofeach sample (25 ul) was measured separately and then the same volume ofeach were combined and measured again. In single sample determinations,an additional equal volume of the buffer was added to maintain the samesample volume as in the combined sample determinations. Results of thetesting are summarized in Table IV.

                  TABLE IV                                                        ______________________________________                                        Single sample determination*                                                                    Combined sample determination*                              (RLU)             (RLU)                                                       DMAE-anti-TSH                                                                           LEAE-anti-TSH                                                                             Theoretical                                                                              Found (%)                                    ______________________________________                                        860,617   1,017,200   1,877,817  1,835,350 (98%)                              173,200     191,820     365,029    362,293 (99%)                              ______________________________________                                         *Each value was the mean of triplicate determinations.                        (RLU -- Relative light units)                                            

The results in Table IV show that the two tracers of different emissionspectra were absolutely non-interfering between each other in theirlight emission. This characteristic further ensures their utility inmulti-analyte binding assays. The LEAE of the preferred method is aN-alkylated benzacridinium compound.

STABILITY of CONJUGATED LBAC'S

LBAC-Anti-TSH conjugates were prepared and tested for their stability inaqueous media. DMAE-anti-TSH conjugate was also tested side by side. Theretention of chemiluminescent activity as a function of temperature atvarious pH's (using citrate-phosphate buffer containing 0.1% BSA) wasmonitored over 7 day period. Proper concentrations of the aboveconjugates (0.8-1.4×10⁶ RLU's/25 ul) were placed in two sets ofdifferent buffers (pH 7.4, 8.0, 8.5, and 9.0). One set was kept at 4°-8°C. as a control, while the other was subjected to 37° C. The bufferedsamples (25 ul) were flashed periodically as described above. Theresults are summarized in Table V.

                  TABLE V                                                         ______________________________________                                        Relative Stability* of Conjugates                                             pH     Compds   1 day    3 days 5 days 7 days                                 ______________________________________                                        7.4    I        93%      98%    91%    95%                                           II       66%      56%    51%    44%                                           III      67%      78%    77%    80%                                           IV       89%      94%    101%   96%                                           V                 66%           46%                                    8.0    I        99%      104%   96%    98%                                           II       106%     106%   80%    83%                                           III      79%      69%    68%    71%                                           IV       102%     114%   140%   138%                                          V                 45%           13%                                    8.5    I        92%      102%   89%    81%                                           II       111%     97%    110%   82%                                           III      81%      76%    78%    86%                                           IV       134%     142%   150%   156%                                   9.0    I        90%      95%    70%    71%                                           III      94%      68%    51%    71%                                           IV       133%     140%   149%   130%                                   ______________________________________                                          Compounds I-V are DMAEanti-TSH, LEAEanti-TSH, 2MeO-LEAE antiTSH,             3EtO-LEAE-anti-Ths, and Nonortho-substituted AEanti-THS, respectively. Th     stability data for Nonortho-substituted AEanti-TSH were equivalent to tha     reported earlier in U.S. Pat. No. 4,745,181.                                  *Relative Stability is defined by expressing the percentage                   chemiluminescent activity of 37° C. samples relative to that of th     corresponding 4-8° C. samples. For example, at pH 8, after 7 days      of storage, the DMAEanti-TSH and LEAEanti-TSH 37° C. samples           retained 87% and 83% activity, respectively, in comparison with the           corresponding 4° C. samples, while the nonortho-substituted            acridinium ester retained only 13% activity in comparison with the            corresponding 4° C. sample.                                       

The stability study summarized in Table V demonstrates that thestabilizing effect of ortho-substitution on the phenoxy ring not onlyapplies to the class of acridinium esters, it also benefits the LBAC'sseries to about the same extent with regard to maintaining theirchemiluminescent activity in aqueous media at or near pH 8 underprolonged heat-stress conditions as required for commercial bindingassay products. Listed in great contrast is the stability data of thenon-ortho-substituted acridinium ester conjugate at pH 8. Anon-ortho-substituted LEAC would likely also have poor stability inaqueous media.

Signal-to-Noise in Binding Assays

LEAE-anti-TSH was employed as tracer in a TSH assay. Performance wasassessed by determining signal-to-noise (S/N) ratio. The performance ofDMAE-antiTSH was also compared side by side. The assay was configured asfollows:

100 microliters of either of the above conjugates was incubated for twohours at room temperature with 100 ul of a TSH standard (Ciba CorningDiagnostics Corp., Medfield, Mass.). Incubations were done separatelywith five standards containing either 0, 0.5, 1.0, 16 or 100 uIu/ml ofTSH. A second incubation was then performed by adding 500 ul of MAGIC®magnetic particle-immobilized with sheep anti-TSH (Ciba CorningDiagnostics Corp.) to the above mixture, then waiting for 30 minutes atroom temperature.

A wash was done first by magnetically separating the particles from thesolution, decanting the solution, then adding 500 ul of water, followedby another magnetic separation. The washed particles were resuspended in100 ul of water. Flashing and counting were done according to theabove-described procedures. The results are provided in Table VI usingratios of the counts with a TSH standard containing TSH versus the zeroTSH standard.

                  TABLE VI                                                        ______________________________________                                                 S/N at various Standards                                             Conjugate  0.4 uIu/ml                                                                             1.0 uIu/ml                                                                              16 uIu/ml                                                                            100 uIu/ml                               ______________________________________                                        DMAE-anti-TSH                                                                            10.0     20.9      202.3  669.4                                    LEAE-anti-TSH                                                                            5.4      8.4       87.4   282.6                                    ______________________________________                                    

The results given in Table VI indicate that LEAE conjugate can beutilized in an immunoassay format to provide a dose-response curve and,therefore, allows the development of useful assays.

DUAL-ANALYTE SIMULTANEOUS IMMUNOASSAY

Instrumentation:

One embodiment of a Dual-PMT Luminometer (DPL) utilized to demonstratethe hardware of DPL includes at least two photo multiplyer tube (PMT)assemblies, an injection pump for Flashing Reagent #2, and a cube-shapelight tight chamber designed for holding a disposable cuvette. Ar twoopposite sides of the chamber, two cylindrical PMT tube assemblies areseparately attached such that light of two different spectral rangesgenerated inside the cuvette can be individually registered by the PMTassemblies. The top of the cuvette-holding chamber is hinged to allowthe cuvette to be manually inserted and removed. In addition, the topalso has a fixed probe attached for the purpose of injecting theFlashing Reagent #2 into the cuvette. Within each PMT assembly anoptical filter selected for particular spectral range, and is insertedbetween the cuvette and the PMT tube.

Alternate embodiments and configurations of DPLs may be designed forsemi-automated and automated detection of two or more chemiluminescentcompounds or conjugates in a test sample. A luminometer as a componenton an automated analyzer is described in EP-A-0 502 638 noted above.

Essential to the discrimination or discernability of two or more emittedlight spectra are the choices of a plurality of optical filters withproper wavelength cutoffs.

Filters of this type are widely available from commercial vendors andmay be modified, i.e. by lamination or specifically manufactured to beincorporated in a PMT assembly for detection and/or quantitation ofspectral signals of the conjugates. Careful selection of filters willenhance the ability to discern emission signals and with appropriatecorrection may allow multiple signals with the emission overlap to bediscerned.

For the purpose of running a simultaneous LH/FSH dual-immunoassay asdisclosed below, a long pass filter (P/N LL-500 of Corion, Holliston,Mass.) and a short pass filter (P/N P70-450 also of Corion) were chosento match with the two different spectral ranges of light generated froma pair of tracers, LEAE-anti-LH and DMAE-anti-FSH, which were preparedin the same manner as described above for LEAE-anti-TSH andDMAE-antiTSH, respectively. The transmittance curves for the two filtersare shown in FIG. 5, panels B and C. The choice of the optical filtersshould take into consideration the requirements on maximal signaltransmittance and minimal signal cross-talk. Optical filters with moredesirable transmittance profile and cut-off may be selected to maximizethe transmission of light emitted from the tracers and/or to fit betterwith the emission spectral ranges of particular chemiluminescentcompounds so as to improve the Percent Cross Talk (PCT) as describedbelow. For example Corion's laminated CS550/CS600 filter (FIG. 5, panelD) was found to be a better replacement for filter P70-450 as the shortpass filter matching with the long pass filter LL-500 for thedetermination of the pair of DMAE and LEAE tracers. Not only was theregistered RLU's for DMAE tracer found to increase by more than 2 foldas a result of this filter's use, the Percentage Cross Talks, as shownin Table VII, were also greatly improved. Furthermore, as more LEAEderivatives with even longer emission maxima were developed, e.g.2-MeO-LEAE, long pass filters such as LL-520 (FIG. 5E) would be a betterchoice than filter LL-500 for enabling further reduction of the PCT.

For system controlling, which generally includes the basic functions ofparameter setting, execution and registration of flashing, signalcorrection as described below as a function of filters used and thechemiluminescent compounds utilized, and data display, a personalcomputer unit containing proper software is utilized and connected tothe DPL.

Percentage Cross-Talks (PCT's) Determination

As mentioned above the two optical filters installed in two separate PMTassemblies on the DPL were intended to gate the emitted lights of twodifferent spectral ranges: the long pass filter is to match with thelonger emission from LEAE tracer and the short pass filter with theshorter emission from DMAE tracer. However, as illustrated by FIGS. 6and 7, because of the minor overlap between the transmittance curves andthe emission spectra of the cross-matching pairs, light signalsgenerated by one tracer can be picked up by the primary PMT intended forit but also in small percentage by the secondary PMT intended for theother tracer, and vice versa. That portion of signal of one tracer, thatcan be registered by the secondary PMT, must be quantitated separatelyin term of percentage for each tracer prior to their use in adual-analyte immunoassay, in order that the apparent RLU's can becorrected and the pure signal of each tracer detected by each PMTassembly be obtained when the two tracers were flashed simultaneously inthe same tube.

Table VII shows the determined PCT's of several pairs of tracers.Anti-FSH-DMAE and anti-LH-LEAE were used in the simultaneous LH/FSHdual-analyte assay described below. Other pairs of tracers were includedto demonstrate that through the selection of acridinium andbenzacridinium compounds of wider separation in their emission maximaand proper choice of optical filters, minimal PCT's ideal formulti-analyte assay can be realized. The PCT's were obtained by dividingthe minor signal from the secondary PMT by the major signal from theprimary PMT in each case, and multiplying the results by 100%.

The concentrations of the samples were randomly selected such that theprimary signals fell in the range of 100,000 to 1,500,000 RLU's per 25ul sample. Each determination was made by sequencially pipeting 25 ul ofone tracer solution, 300 ul of Flashing Reagent #1 into the cuvette,vortexing the resulting solution briefly, inserting the cuvette into thePMT housing, and performing the flashing by injecting 300 ul of FlashingReagent #2 through the key-board control.

                  TABLE VII                                                       ______________________________________                                        Determination of Percent Cross-Talk (PCT)                                               Long Pass Signals                                                                         Short Pass Signals                                      Sample    (RLU's)     (RLU's)      PCT (%)                                    ______________________________________                                        SET (I)*:                                                                     LH tracer 538         688                                                     diluent   444         396                                                     Anti-LH-LEAE                                                                            6.25 E5     4.18 E4           6.7                                             5.78 E5     3.91 E4           6.8                                             6.19 E5     4.18 E4           6.8                                                                      Aver.                                                                              6.8                                   FSH tracer                                                                              242         288                                                     diluent   268         340                                                     Anti-FSH-DMAE                                                                           2.32 E4     1.59 E5           14.5                                            2.42 E4     1.65 E5           14.7                                            2.60 E4     1.75 E5           14.8                                                                     Aver.                                                                              14.7                                  SET (II) :                                                                    Anti-LH-LEAE                                                                            1.27 E6     5.52 E4           4.4                                             1.25 E6     5.40 E4           4.3                                                                      Ave. 4.4                                   Anti-FSH-DMAE                                                                           4.62 E4     4.28 E5           10.8                                            4.60 E4     4.23 E5           10.9                                                                     Ave. 10.9                                  SET (III).sup.+ :                                                             Buffer.sup.˜                                                                      348         278                                                               350         284                                                     Anti-TSH-2-                                                                             9.90 E5     1.29 E4           1.3                                   MeO-LEAE  9.88 E5     1.29 E4           1.3                                                                      Ave. 1.3                                   Anti-FSH-DMAE                                                                           1.17 E4     3.77 E5           3.1                                             1.17 E4     3.81 E5           3.1                                                                      Ave. 3.1                                   SET (IV).sup.# :                                                              Buffer.sup.˜                                                                      187         608                                                               182         429                                                     Anti-TSH-2-                                                                             2.12 E5     6.26 E3           2.9                                   MeO-LEAE  2.0 E5      5.95 E3           2.9                                                                      Aver.                                                                              2.9                                   Anti-TSH-DMAE                                                                           2.93 E4     5.39 E5           5.4                                             3.12 E4     5.83 E5           5.4                                                                      Aver.                                                                              5.4                                   ______________________________________                                         *Optical filters mounted on the DPL: LL500 & P70450.                           Optical filters mounted on the DPL: LL500 & Laminated CS550/CS-600.          .sup.+ Optical filters mounted on the DPL: LL520 & Laminated CS550/CS-600     .sup.# Optical filters mounted on the DPL: LL520 & P70450.                    .sup.˜ The buffer was 10 mM PBS/0.1% BSA/0.05% NaN.sub.3, pH 8.0.  

The constancy of the PCT over a wide range of RLU's is critical in themulti-analyte assay signal correction. Table VIII shows that when thelaminated CS550/CS600 filter and LL520 filter were used to gate theshort pass and long pass signals, respectively, the PCT foranti-TSH-DMAE has the mean of 2.96% with standard deviation of 0.16%over RLU range of 10,000 to 7,000,000 counts or broader, while the PCTfor anti-CKMB-LEAE has the mean of 4.79% with standard deviation of0.23% over RLU range of 50,000 to 7,000,000 counts or broader.

                  TABLE VIII                                                      ______________________________________                                        Constancy of Percent Cross-Talk                                                           Short     Long                                                                Pass Signal                                                                             Pass Signal                                                                              PCT  Mean/SD                                 Sample      (RLU's)   (RLU's)    (%)  (%)                                     ______________________________________                                        Anti-TSH-DMAE                                                                             6,689,796 217,010    3.24                                                     6,545,872 212,956    3.25                                                     6,645,674 210,262    3.16                                                     1,469,500 43,710     2.97                                                     1,469,770 45,004     3.06                                                     1,450,042 43,492     3.00                                                     303,944   8,922      2.81*                                                    302,876   8,788      2.78*                                                    287,632   8,928      2.98*                                                    59,468    2,106      2.95*                                                    58,816    2,072      2.93*                                                    60,314    1,996      2.73*                                                    12,298    692        2.77*                                                    11,956    692        2.86*                                                    12,420    716        2.96*                                                                              2.96/0.16                               Buffer Diluent                                                                            738       496                                                                 702       320                                                     Anti-CKMB-LEAE                                                                            320,758   6,663,484  4.81                                                     315,344   6,497,756  4.85                                                     320,224   6,528,242  4.91                                                     61,374    1,350,584  4.54                                                     61,514    1,329,548  4.63                                                     60,036    1,330,152  4.51                                                     13,044    264,526    4.71*                                                    11,968    244,106    4.67*                                                    12,324    245,542    4.78*                                                    3,312     56,120     4.84*                                                    3,586     54,906     5.45*                                                    3,220     53,928     4.87*                                                                              4.79/0.23                               Buffer Diluent                                                                            652       372                                                                 622       346                                                     ______________________________________                                         *Correction was made in consideration of the additional signal contribute     by the buffer and system noise.                                          

Equations for Correcting the Apparent RLU's due to Cross-Talks inDual-Tracer Determination

When DMAE and LEAE derivatives or tracers are mixed and flashedsimultaneously, the observed long and short pass signals can be brokendown as follows:

    S(s)=S(DMAE)+S'(LEAE)+b1                                   (1)

    S(l)=S(LEAE)+S'(DMAE)+b2                                   (2)

Where, S(s) and S(l) are the observed short and long pass signals,respectively; S(DMAE) and S(LEAE) are the portions of signals due toDMAE and LEAE in the observed short and long pass signals, respectively.They will also be referred to as the corrected DMAE and LEAE signals;S'(DMAE) and S'(LEAE) are portions of the long and short pass signalsdue to DMAE and LEAE cross-talking, respectively; b1 and b2 are thecombined signals due to assay components and system noise in the absenceof DMAE and LEAE tracers, respectively.

Since the PCT's (represented by k1 and k2 below) are constants for anyparticular DMAE and LEAE tracers, there exist the followingrelationships:

    S'(DMAE)=k1×S(DMAE)                                  (3)

    S'(LEAE)=k2×S(LEAE)                                  (4)

Where k1, k2 are the PCT's for the DMAE and LEAE tracers, respectively.

Substitute equation (4) into (1):

    S(s)=S(DMAE)+k2×S(LEAE)+b1

    or S(DMAE)=S(s)-k2×S(LEAE)-b1                        (5)

Substitute equations (5) into (3) and (3) into (2): ##EQU1##Rearranging: ##EQU2## Equations (5) and (6) will yield the correctedshort pass signal due to DMAE tracer and long pass signal due to LEAEtracer, respectively. For the purpose of demonstrating the feasibilityof conducting a simultaneous LH/FSH dual-analyte assay, thedetermination of the combined matrix and system noises, b1 and b2 wasfound not to be significant. They were therefore both assigned a 0 valuein the signal corrections for the following examples of the dual-analyteassays.

Simultaneous Immunoassay for Luteinizing Hormone (LH) and FollicleStimulating Hormone (FSH)

One objective of the invention is to provide a method for simultaneouslydetecting and/or quantitating two or more substances or analytes in asingle sample through the utilization of two different chemiluminescentlabels or conjugates.

In an example of one embodiment, the assay system utilizes a DMAElabelled FSH antibody and a LEAE labelled LH antibody. The followingexamples demonstrate that LH and FSH standard curves and sample recoveryare identical within the limits of experimental error when each analyteis assayed as a single analyte by introduction of one chemiluminescenttracer into the assay system, or in a dual analyte system which employstwo chemiluminescent tracers. The examples further show that tracersprepared from a pair of a DMAE and a LEAC can be utilized in asimultaneous assay of two substances for which a corresponding bindingpartner, e.g. antibody, is available.

EXAMPLE 15 Single FSH assay using Dual-Analyte Immunoassay System

The Magic Lite FSH kit components and protocol (Ciba CorningDiagnostics) were modified such that the assay could be performed as asingle or dual analyte assay depending on the tracer selection. A solidphase consisting of paramagnetic particles (PMP) coupled to anti-FSHantibodies and PMP coupled to anti-LH antibodies was prepared byremoving the buffer diluent from the Magic Lite FSH kit solid phase andresuspending these particles in Magic Lite LH kit solid phase (CibaCorning Diagnostics Corp.). The kit tracer, anti-FSH-DMAE, was diluted1:2 in Magic Lite LH kit tracer buffer. Standards for calibrationcontained both FSH and LH. Standards were prepared by spiking knownconcentrations of purified human FSH and human LH into a horse serumbasepool. Nominal standard values were 0, 0.9, 2.2, 4.4, 8.8, 21.9,43.8, 87.5, 140.0, 201.0 mIu/ml of FSH. Nominal LH concentrations were0, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 160.0, 230.0 mIu/ml LH.Samples for, analysis were prepared by spiking a human serum pool withvarying concentrations of both purified human FSH and human LH.Additionally, serum based multi-constituent calibrators containing humanFSH and human LH were used as samples.

To perform the assay, 50 ul of each standard or sample and 200 ul ofdiluted FSH tracer were vortex mixed and incubated for 30 minutes atroom temperature. 500 ul of the combined anti-FSH/anti-LH solid phasewas added, vortex mixed and incubated for 30 minutes at roomtemperature. The reacted solid phase was magnetically separated for 3minutes in a Magic Lite rack (Ciba Corning Diagnostics Corp.), seeEuropean Patent 136126, and the supernatant decanted. The reacted solidphase was next washed with 1.0 ml of distilled water, separated for 3minutes. The supernatant was decanted, and 100 ul of distilled wateradded. Each sample was manually transferred to a cuvette, and countedfor 5 seconds on the DPL described above. The results (in RLU's)obtained from the short pass (DMAE) channel were used to calculate FSHconcentration in each sample. Concentrations were calculated by using10-point calibration with a spline data reduction routine. Each standardand sample was assayed in replicates of three. RLU's and %CVC for thisassay are shown in Table IX under the heading FSH single-analyte assay.FSH sample recovery is shown in Table X under the heading FSHsingle-analyte assay. The FSH standard curve presented as %B/Bmax vs logFSH concentration is shown in FIG. 8 labelled as FSH single-analyteassay.

EXAMPLE 16 Single LH Assay using Dual-analyte Immunoassay System

The solid phase reagent, standards, and samples described in Example 15were used to perform an LH assay. The anti-FSH-DMAE tracer was replacedwith an anti-LH-LEAE tracer which was diluted 1:2 in Magic Lite FSH kittracer diluent. The assay methodology described in Example 13 wasapplied to this assay, except that the RLU results obtained from theLong pass (LEAE) channel were used to calculate LH sampleconcentrations.

The assay was calibrated using nine of the standards described inExample 15, excluding the 1.0 mIu/ml LH standard. Results for this assayare shown in Table XI and Table XII under the heading LH single-analyteassay. The standard curve is shown in FIG. 9 labelled as LHsingle-analyte assay.

EXAMPLE 17 Simultaneous LH/FSH Assay using Dual-analyte

Immunoassay System

Solid phase reagent, standards, and samples described in Example 15 and16 were used to perform a dual label LH/FSH assay in a single tube. Thetracer consisted of the Magic Lite FSH kit tracer, anti-FSH-DMAE diluted1:2 in the anti-LH-LEAE tracer. The assay methodology was the same asthat described in Example 15. The raw RLU's from each channel wasmathematically corrected for cross-talk prior to concentrationcalculations. Corrected RLU's and concentrations resulting from thesecorrected RLU's are shown in Tables IX-XII, and are labelled asdual-analyte assay. Mean sample recovery for single analyte vs. dualanalyte assays are compared by t-test in Tables X and XII. The FSH andLH standard curves are shown in FIGS. 8 and 9 and labelled as FSH and LHdual-analyte assay, respectively.

Assays and Assay Formats

The present invention relates to chemiluminescent compounds and moreparticularly, the use of two or more chemiluminescent conjugates tosimultaneously detect two or more substances in a test sample. Thedisclosure teaches the use of benzacridinium compounds and preferablyN-alkylated benzacridinium compounds in such assays.

A test substance includes any component(s) or analytes sought to bedetected and/or quantitated in a test sample, including but not limitedto, more than one component of a single structure, e.g. more than oneportion of a nucleic acid sequence or different loci of a chromosome,genome or molecule, where the components or analytes may be ofbiological or industrial origin, such as nucleic acids, proteins,ligands, haptens or other materials or compounds to which an appropriateassay method can be formatted. It is understood that the test sampleand/or substance may need to be pretreated to render it assayable by atest method. The test substances and quantities thereof sought to bedetected may limit the types of assays which can be performed becauseof, for example, sensitivity concerns, but not the use ofchemiluminescent labels for detection. Various internal standards orcontrols may be added to a test sample for detection and/or quantitationto asess the performance of the assay. Diagnostic assays exemplified byimmunoassays, hybridization assays and amplification assays haveincreasingly incorporated chemiluminescent labels in their formats.Designs and formats of such assays are well known by those skilled inthe art and extensively published in the technical and patentliterature, for example, an assay format may require the seperation of areaction product or unreacted agent to a transfer tube for detectionand/or quantitation. Such separation techniques may be useful forcompetitive assays, solid phase assays or to limit interferents.

In one embodiment of the invention, two or more chemiluminescentconjugates are utilized as labels in an amplification assay.Representative amplification assays include but should not be limited topolymerase chain reaction (PCR), autocatalytic replication ofrecombinant RNA and amplification of midivariant DNA or RNA. See EP-A-0481 704 (priority U.S. Ser. No. 598,269 (Oct. 16, 1990)abandoned) whichis commonly assigned and incorporated herein by reference. Such methods,as taught in the technical and patent may be made adaptable toincorporate chemiluminescent labels, and particularly two or morechemiluminescent labels for detection of target sequences of interest.The advantage of using a multi-label method is to detect and/orquantitate a plurality of target sequences or one or more targetsequences and an internal standard. An example of such a method includesproviding a test sample suspected of containing one or more targetsequences, amplifying the target sequences, providing at least twochemiluminescent conjugates, each chemiluminescent conjugate beingassociated with a target sequence(s) and simultaneously detecting and/orquantifying amplified target sequences by emissions of at least twochemiluminescent conjugates. In another step of this method an internalreference, control or control system may be added to the assay to insureassay performance and results. The internal reference may be amplifiedas well as the target sequences.

The use of chemiluminescent labels for such assays serves to demonstratethe utility of this invention.

The chemiluminescent compounds of this invention are adapted to bepackaged in kit form for commercial sale. The chemiluminescent labels ofthese kits may be conjugated to appropriate substances or materialswhich are specific to the substances sought to be detected in the testsamples. Appropriate functional groups may be added to thechemiluminescent compounds for use in various assays and otherapplications. Examples of assays for which the methods of the presentinvention may be utilized include but should not be limited to: assaysincluding at least two antibodies of different specifities; assaysincluding at least two antigens; assays including at least one antigenand at least one antibody; and assays for molecules indicative ofcancer, infectious diseases, genetic abnormalities, genetic disposition,genetic assessment and to monitor medicinal therapy.

It is to be understood that various other modifications will be apparentto and can readily be made by those skilled in the art, given thedisclosure herein, without departing from the Scope and materials ofthis invention. It is not, however, intended that the scope of theclaims appended hereto be limited to the description as set forthherein, but rather that the claims be construed as encompassing allfeatures of patentable novelty which reside in the present invention,including all features which would be treated as equivalents thereof bythose skilled in the art to which the invention pertains. It is alsonoted that the examples given therein are intended to illustrate, andnot to limit the invention.

                  TABLE IX                                                        ______________________________________                                        MEAN RLU's and % CVC FOR MODIFIED MAGIC LITE FSH                              ASSAYS                                                                                  FSH SINGLE  FSH RESULTS DUAL                                                  ANALYTE ASSAY                                                                             ANALYTE ASSAY*                                          SAMPLE (VALUE)                                                                            MEAN RLU  % CVC   MEAN RLU % CVC                                  ______________________________________                                        S1 (0)      1336      4.3     610      6.7                                    S2 (0.9)    1891      5       1309     14.4                                   S3 (2.2)    3202      10.9    2375     6.7                                    S4 (4.4)    5155      9.4     4189     3.9                                    S5 (8.8)    9005      2.6     7702     2.4                                    S6 (21.9)   19637     5.5     16942    5.2                                    S7 (43.8)   35491     0.7     30563    4                                      S8 (87.5)   56844     1.7     41969    5                                      S9 (140.0)  74850     0.9     66499    0.5                                    S10 (201.0) 87531     0.8     77003    0.5                                    SAMPLE 1    9962      7.3     8417     2.8                                    SAMPLE 2    16779     4.9     14777    0.8                                    SAMPLE 3    29683     13      28721    4.5                                    SAMPLE 4    62549     2.2     55878    2.4                                    SAMPLE 5    93019     1.1     84182    3.3                                    SAMPLE 6    103290    3       93297    1.2                                    LOW MULTI-CAL                                                                             1756      9       1160     9.8                                    HIGH MULTI-CAL                                                                            80308     2.4     71082    0.6                                    TOTAL COUNTS                                                                              564200            571392                                          ______________________________________                                         *corrected RLU                                                           

                                      TABLE X                                     __________________________________________________________________________    FSH SAMPLE RECOVERY: SINGLE ANALYTE ASSAY VS. DUAL ANALYTE ASSAY                        FSH SINGLE                                                                             FSH DUAL      CRITICAL                                               ANALYTE ASSAY                                                                          ANALYTE ASSAY T-VALUE                                      SAMPLE    MIU/ML                                                                             % CVD                                                                             MIU/ML                                                                             % CVD                                                                             T-VALUE                                                                            95% C.I.                                     __________________________________________________________________________    SAMPLE 1  9.923                                                                              8.6 9.737                                                                              3.2 0.36 +/-4.30                                      SAMPLE 2  18.291                                                                             5.7 18.668                                                                             1   -0.62                                                                              +/-4.30                                      SAMPLE 3  35.235                                                                             15.1                                                                              40.708                                                                             5.3 -1.65                                                                              +/-4.30                                      SAMPLE 4  101.951                                                                            3.5 98.877                                                                             4.2 0.98 +/-3.18                                      SAMPLE 5  >>       >>                                                         SAMPLE 6  >>       >>                                                         MULTI-CAL LOW                                                                           0.693                                                                              30.8                                                                              0.697                                                                              20.1                                                                              -0.2 +/-3.18                                      MULTI-CAL HIGH                                                                          163.132                                                                            5.4 163.812                                                                            1.4 -0.13                                                                              +/-4.30                                      __________________________________________________________________________

                  TABLE XI                                                        ______________________________________                                        MEAN RLU'S AND % CVC FOR MODIFIED MAGIC LITE LH                               ASSAYS                                                                                  LH SINGLE   LH RESULTS DUAL                                                   ANALYTE ASSAY                                                                             ANALYTE ASSAY*                                          SAMPLE (VALUE)                                                                            MEAN RLU  % CVC   MEAN RLU % CVC                                  ______________________________________                                        S1 (0)      17,883    14.2    18,477   10.6                                   S3 (2.5)    24,310    1.9     22,818   2.7                                    S4 (5.0)    27,007    6.5     25,118   11.9                                   S5 (10.0)   34,713    11.6    28,934   6.2                                    S6 (25.0)   42,440    4.6     41,543   5.3                                    S7 (50.0)   65,787    2.9     71,140   6.2                                    S8 (100.0)  115,760   6.8     113,694  1.9                                    S9 (160.0)  161,767   4.4     164,225  4.4                                    S10 (230.0) 223,569   4.8     205,347  4.5                                    SAMPLE 4    42,157    2.8     40,519   8.2                                    SAMPLE 5    82,934    4.3     83,592   5.5                                    SAMPLE 6    110,502   2.8     113,285  1.9                                    MULTI-CAL HIGH                                                                            86,189    3.5     90,451   2.9                                    TOTAL COUNTS                                                                              2,250,526         2,322,672                                       ______________________________________                                         *CORRECTED RLU                                                           

                                      TABLE XII                                   __________________________________________________________________________    LH SAMPLE RECOVERY: SINGLE ANALYTE ASSAY VS. DUAL ASSAY                                 LH SINGLE                                                                              LH RESULTS DUAL                                                                              CRITICAL                                              ANALYTE ASSAY                                                                          ANALYTE ASSAY* T-VALUE                                     SAMPLE    MIU/ML                                                                             % CVD                                                                             MIU/ML                                                                             % CVD                                                                              T-VALUE                                                                            95% C.I.                                    __________________________________________________________________________    SAMPLE 4  21.347                                                                             7   22.342                                                                             15.3 -0.46                                                                              +/-4.30                                     SAMPLE 5  67.392                                                                             5.7 64.829                                                                             7.5  0.72 +/-3.18                                     SAMPLE 6  97.375                                                                             3.6 97.984                                                                             2.5  -0.25                                                                              +/-3.18                                     MULTI-CAL HIGH                                                                          70.906                                                                             4.5 72.153                                                                             4    -0.5 +/-3.18                                     __________________________________________________________________________

The invention having thus been described, what is claimed as new anddesired to secure by Letters Patent is:
 1. A chemiluminescent compoundof the formula: ##STR13## where W is carbon; alternatively C₇, W, C₉ orC₁₀ is replaced with --N═;or alternatively W is omitted and C₇ isconnected to C₉ end optionally C₇, C₉ or C₁₀ is replaced with --O--,--S--, --NH-- or --NR--; R_(2a), R_(2b), R_(2c), R_(2d). R_(3a), R_(3b),R_(3c), R_(3d), R₉ and R₁₀ are identical or different groups selectedfrom hydrogen, substituted aryl, unsubstituted aryl, halide, amino,hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --OR, --SR, --SSR,--C(O)R, --C(O)OR, --C(O)NHR, or NHC(O)R; R_(2c) and R_(2b) areoptionally a fused aromatic ring with or without heteroatoms; A⁻ is acounter ion; R₁ is alkyl, alkenyl, alkynyl, or aralkyl, optionallycomprising up to 20 heteroatoms; Y is a branched or straight chained.halogenated or unhalogenated, alkyl optionally comprising up to 20carbon atoms, or a polysubstituted aryl moiety of the formula: ##STR14##R₄ and R₈ are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol or amido, R₅and R₇ are identical or different groups selected from hydrogen,substituted aryl, unsubstituted aryl, halide, amino, hydroxyl, nitro,sulfonate, --R, --CN, --COOH, --SCN, --OR, --SR, --SSR, --C(O)R,--C(O)OR, --C(O)NHR, or NHC(O)R; R₆ =--R₁₁ --R₁₂, where R₁₁ is notrequired but optionally is a branched or straight-chained alkyl, or asubstituted or unsubstituted aryl or aralkyl containing optionally up to20 heteroatoms; and R₁₂ is a leaving group or an electrophilicfunctional group attached with a leaving group or --Q--R--Nu,--Q--R(I)_(n) Nu, --Q--Nu, --R--Nu or Nu, where n is a number of atleast 1, Nu is a nucleophilic group, Q is a functional linkage, I is anionic or ionizable group; R₅ and R₆, and R₆ and R₇ are interchangeable;R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally upto 20 heteroatoms; and X is a heteroatom selected from nitrogen, oxygenor sulfur, such that when X is oxygen or sulfur Z is omitted, when X isnitrogen Z is --SO₂ --Y', and Y' is equal to Y, where the substituentsto Y and Y' do not have to be the same; wherein said compound, uponchemical treatment, emits blue-green, green, yellow, orange orred-orange light.
 2. A chemiluminescent compound of the formula:##STR15## where W is carbon; alternatively C₇, W, C₉ or C₁₀ is replacedwith --N═;or alternatively W is omitted and C₇ is connected to C₉ andoptionally C₇, C₉ or C₁₀ is replaced with --O--, --S--, --NH-- or--NR--; R_(2a), R_(2b), R_(2c), R_(2d), R_(3a), R_(3b), R_(3c), R_(3d),R₉ and R₁₀ are identical or different groups selected from hydrogen,substituted aryl, unsubstituted aryl, halide, amino, hydroxyl, nitro,sulfonate, --R, --CN, --COOH, --SCN, --OR, --SR, --SSR, --C(O)R,--C(O)OR, --C(O)NHR, or NHC(O)R; R_(2c) and R_(2b) are optionally afused aromatic ring with or without heteroatoms; A⁻ is a counter ion; R₁is alkyl, alkenyl, alkynyl, or aralkyl, optionally comprising up to 20heteroatoms; Y is a branched or straight chained, halogenated orunhalogenated, alkyl optionally comprising up to 20 carbon atoms, or apolysubstituted aryl moiety of the formula: ##STR16## R₄ and R₈ arealkyl, alkenyl, alkynyl, alkoxyl, alkylthiol or amido, R₅ and R₇ areidentical or different groups selected from hydrogen, substituted aryl,unsubstituted aryl, halide, amino, hydroxyl, nitro, sulfonate, --R,--CN, --COOH, --SCN, --OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, orNHC(O)R; R₆ =--R₁₁ --R₁₂, where R₁₁ is not required but optionally is abranched or straight-chained alkyl, or a substituted or unsubstitutedaryl or aralkyl containing optionally up to 20 heteroatoms; and R₁₂ is aleaving group or an electrophilic functional group attached with aleaving group or --Q--R--Nu, --Q--R(I)_(n) Nu, --Q--Nu, --R--Nu or Nu,where n is a number of at least 1, Nu is a nucleophilic group, Q is afunctional linkage, I is an ionic or ionizable group; R₅ and R₆, and R₆and R₇ are interchangeable; R is alkyl, alkenyl, alkynyl, aryl oraralkyl containing optionally up to 20 heteroatoms; and X is aheteroatom selected from nitrogen, oxygen or sulfur, such that when X isoxygen or sulfur Z is omitted, when X is nitrogen Z is --SO₂ --Y', andY' is equal W Y, where the substituents to Y and Y' do not have to bethe same; wherein said compound, upon chemical treatment, emitsblue-green, green, yellow, orange and red-orange light.
 3. Achemiluminescent compound as recited in claim 2, wherein said compoundhas an emission spectra peak or maximum greater than about 508 nm.
 4. Achemiluminescent compound as recited in claim 2, wherein R₁ is alkyl. 5.A luminescent compound of the formula: ##STR17## where W is carbon;alternatively C₇, W, C₉ or C₁₀ is replaced with --N═;or alternatively Wis omitted and C₇ is connected to C₉ and optionally C₇, C₉ or C₁₀ isreplaced with --O--, --S--, --NH-- or --NR--; R_(2a), R_(2b), R_(2c),R_(2d), R_(3a), R_(3b), R_(3c), R_(3d), R₉ and R₁₀ are identical ordifferent groups selected from hydrogen, substituted aryl, unsubstitutedaryl, halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --COOH,--SCN, --OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or NHC(O)R;R_(2c) and R_(2b) are optionally a fused aromatic ring with or withoutheteroatoms; A⁻ is a counter ion; R₁ is alkyl, alkenyl, alkynyl, oraralkyl, optionally comprising up to 20 heteroatoms; X is oxygen orsulfur and Y is a polysubstituted aryl moiety of the formula: ##STR18##R₄ and R₈ are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol or amido, R₅and R₇ are identical or different groups selected from hydrogen,substituted aryl, unsubstituted aryl, halide, amino, hydroxyl, nitro,sulfonate, --R, --CN, --COOH, --SCN, --OR, --SR, --SSR, --C(O)R,--C(O)OR, --C(O)NHR, or NHC(O)R; R₆ =--R₁₁ --R₁₂, where R₁₁ is notrequired but optionally is a branched or straight-chained alkyl, or asubstituted or unsubstituted aryl or aralkyl containing optionally up to20 heteroatoms; and R₁₂ is a leaving group or an electrophilicfunctional group attached with a leaving group or --Q--R--Nu,--Q--R(I)_(n) Nu, --Q--Nu, --R--Nu or Nu, where n is a number of atleast 1, Nu is a nucleophilic group, Q is a functional linkage, I is anionic or ionizable group; R₅ and R₆, and R₆ and R₇ are interchangeable;and R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionallyup to 20 heteroatoms.
 6. A luminescent compound as recited in claim 5,wherein said compound is conjugated with a binding partner for use intest assays.
 7. A luminescent compound as recited in claim 5, wherein A⁻is selected from the group consisting of CH₃ SO₄ --, FSO₃ --, CF₃ SO₃--, C₄ F₉ SO₃ --, CH₃ C₆ H₄ SO₃ -- and halide.
 8. A chemiluminescentcompound selected from the group consisting of:(a)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (LEAE-Bz); (b)(2',6'-dimethyl-4-succinimidyloxycarbonyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (LEAE-NHS); (c)(4'-benzyloxycarbonyl-2',6'-diisopropyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (DIP-LEAE-Bz); (d)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 3-ethoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (3-EtO-LEAE-Bz); (e)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-(N,N-diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridinium-12-carboxylate difluorosulfonate (3-QAE-LEAE-Bz); (f)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (2-MeO-LEAE-Bz); (g)(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl5-methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylatedifluorosulfonate (2-QAE-LEAE-NHS); (h)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 5-(3-sulfopropyl)-benzb!acridinium-12-carboxylate (NSP-LEAE-Bz); (i)(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl2-methoxy-5-(2-sulfoethyl)-benz b!acridinium-12-carboxylate(2-MeO-NSE-LEAE-NHS); and (j)(2',6'-dimethyl-4'-(2-methoxyiminoethyl)/phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate dichloride (2-MeO-LEAE-Imidate).
 9. Anintermediate compound for the synthesis of a luminescent compound of theformula: ##STR19## where: W is --N═; orW is carbon and C₇, C₉, or C₁₀ is--N═; or W is omitted, C₇ is connected to C₉, and C₇, C₉, or C₁₀ is--O--, --S--, --NH--, or --NR--; R_(2a), R_(2b), R_(2c), R_(2d), R_(3a),R_(3b), R_(3c), R_(3d), R₉ and R₁₀ are identical or different groupsselected from hydrogen, substituted aryl, unsubstituted aryl, halide,amino, hydroxyl, nitro, sulfonate, --R, --CN,--COOH, --SCN, --OR, --SR,--SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R; R_(2c) and R_(2b) areoptionally a fused aromatic ring with or without heteroatoms; and R isselected from alkyl, alkenyl, alkynyl, aryl or aralkyl containingoptionally up to 20 heteroatoms.
 10. An intermediate compound for thesynthesis of a luminescent compound of the formula: ##STR20## where W iscarbon; alternatively C₇, W, C₉ or C₁₀ is replaced with --N═;oralternatively W is omitted and C₇ is connected to C₉ and optionally C₇,C₉ or C₁₀ is replaced with --O--, --S--, --NH-- or --NR--; R_(2a),R_(2b), R_(2c), R_(2d), R_(3a), R_(3b), R_(3c), R_(3d), R₉ and R₁₀ areidentical or different groups selected from hydrogen, substituted aryl,unsubstituted aryl, halide, amino, hydroxyl, nitro, sulfonate, --R,--CN, --COOH, --SCN, --OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, orNHC(O)R; R_(2c) and R_(2b) are optionally a fused aromatic ring with orwithout heteroatoms; X is oxygen or sulfur and Y is a polysubstitutedaryl moiety of the formula: ##STR21## R₄ and R₈ are alkyl, alkenyl,alkynyl, alkoxyl, alkylthiol or amido, R₅ and R₇ are identical ordifferent groups selected from hydrogen, substituted aryl, unsubstitutedaryl, halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --COOH,--SCN, --OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or NHC(O)R; R₆=--R₁₁ --R₁₂, where R₁₁ is not required but optionally is a branched orstraight-chained alkyl, or a substituted or unsubstituted aryl oraralkyl containing optionally up to 20 heteroatoms; and R₁₂ is a leavinggroup or an electrophilic functional group attached with a leaving groupor --Q--R--Nu, --Q--R(I)_(n) Nu, --Q--Nu, --R--Nu or Nu, where n is anumber of at least 1, Nu is a nucleophilic group, Q is a functionallinkage, I is an ionic or ionizable group; R₅ and R₆, and R₆ and R₇ areinterchangeable; and R is alkyl, alkenyl, alkynyl, aryl or aralkylcontaining optionally up to 20 heteroatoms.
 11. A method for making achemiluminescent compound selected from the group consisting of:(a)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (LEAE-Bz); (b)(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (LEAE-NHS); (c)(4'-benzyloxycarbonyl-2',6'-diisopropyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (DIP-LEAE-Bz); (d)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 3-ethoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (3-EtO-LEAE-Bz); (e)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-(N,N-diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridinium-12-carboxylate difluorosulfonate (3-QAE-LEAE-Bz); (f)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (2-MeO-LEAE-Bz); (g)(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl5-methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylatedifluorosulfonate (2-QAE-LEAE--NHS); (h)(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 5-(3-sulfopropyl)-benzb!acridinium-12-carboxylate (NSP-LEAE-Bz); (i)(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl2-methoxy-5-(2-sulfoethyl)-benz b!acridinium-12-carboxylate(2-MeO-NSE-LEAE-NHS); (j){2',6'-dimethyl-4'-(2-methoxyiminoethyl)}phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate dichloride (2-MeO-LEAE-Imidate);said methodcomprising: in the case of (4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl5-methyl-benz b!acridinium-12-carboxylate fluorosulfonate (LEAE-Bz):(1)reacting 3-hydroxy-2-naphthoic acid and aniline to produce3-anilino-2-naphthoic acid; (2) treating 3-anilino-2-naphthoic acid withphosphorous oxychloride to produce 12-chloro-benz b!acridine; (3)treating 12-chloro-benz b!acridine with potassium cyanide and copper (I)cyanide to produce 12-cyano-benz b!acridine; (4) treating 12-cyano-benzb!acridine with tetrabutylammonium bromide and sulfuric acid, then witha sodium hydroxide solution followed by concentrated HCl to produce benzb!acridine-12-carboxylic acid hydrochloride; (5) treating benzb!acridine-12-carboxylic acid hydrochloride with p-toluenesulfonylchloride and benzyl 3,5-dimethyl-4-hydroxybenzoate to produce(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl benzb!acridine-12-carboxylate; and (6) treating(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl benzb!acridine-12-carboxylate with methyl fluorosulfonate to produceLEAE-Bz; in the case of(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl 5-methyl-benzb!acridinium-12-carboxylate Fluorosulfonate (LEAE-NHS):(1) treating(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl benzb!acridine-12-carboxylate with hydrogen bromide in acetic acid toproduce (4'-carboxy-2',6'-dimethyl)phenyl benz b!acridine-12-carboxylatehydrobromide; (2) treating (4'-carboxy-2',6'-dimethyl)phenyl benzb!acridine-12-carboxylate hydrobromide with dicyclohexylcarbodiimide andN-hydroxysuccinimide to produce(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl benzb!acridine-12-carboxylate; and (3) treating(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl benzb!acridine-12-carboxylate with methyl fluorosulfonate to produceLEAE-NHS; in the case of (4'-benzyloxycarbonyl-2',6'-diisopropyl)phenyl5-methyl-benz b!acridinium-12-carboxylate fluorosulfonate(DIP-LEAE-Bz):(1) treating 2,6-diisopropylphenol with sodium methoxideand carbon dioxide to produce 3,5-diisopropyl-4-hydroxybenzoic acid; (2)esterifying 3,5-diisopropyl-4-hydroxybenzoic acid by forming first thepotassium salt, then treating the salt with a crown ether andbenzylbromide to produce benzyl 3,5-diisopropyl-4-hydroxybenzoate; (3)treating benz b!acridine-12-carboxylic acid hydrochloride withp-toluenesulfonyl chloride and benzyl 3,5-diisopropyl-4-hydroxybenzoateto produce (4'-benzyloxycarbonyl-2',6'-diisopropyl)phenyl benzb!acridine-12-carboxylate; and (4) treating(4'-benzyloxycarbonyl-2',6'-diisopropyl)phenyl benzb!acridine-12-carboxylate with methyl fluorosulfonate to produceDIP-LEAE-Bz; in the case of (4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-ethoxy-5-methyl-benz b!acridinium-12-carboxylate fluorosulfonate(3-EtO-LEAE-Bz):(1) treating benzo 5,6!isatin with potassium hydroxideand resorcinol to produce 3-hydroxy-benz b!acridine-12-carboxylic acid;(2) treating 3-hydroxy-benz b!acridine-12-carboxylic acid with cesiumcarbonate and bromoethane to produce ethyl 3-ethoxy-benzb!acridinium-12-carboxylate; (3) treating ethyl 3-ethoxy-benzb!acridinium-12-carboxylate with sodium hydroxide in ethanol andacidifying to produce 3-ethoxy-benz b!acridinium-12-carboxylic acidhydrochloride; (4) treating 3-ethoxy-benz b!acridinium-12-carboxylicacid hydrochloride with p-toluenesulfonyl chloride, benzyl3,5-dimethyl-4-hydroxybenzoate, and N,N'-dimethylamino-pyridine toproduce (4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 3-ethoxyobenzb!acridinium-12-carboxylate; and (5) treating(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 3-ethoxy-benzb!acridinium-12-carboxylate with methyl fluorosulfonate to produce3-EtO-LEAE-Bz; in the case of(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-(N,N-diethyl-N-methyl-ammonium)ethoxy-5-methyl-benzb!acridinium-12-carboxylate difluorosulfonate (3-QAE-LEAE-Bz):(1)treating 3-hydroxy-benz b!acridine-12-carboxylic acid with cesiumcarbonate and diethylaminoethyl bromide hydrobromide to produceN,N-diethylaminoethyl 3-(N,N-diethylamino)ethoxy-benzb!acridine-12-carboxylate; (2) treating N, N-diethylaminoethyl3-(N,N-diethylamino) ethoxy-benz b!acridine-12-carboxylate with sodiumhydroxide in methanol and acidifying to produce3-(N,N-diethylamino)ethoxy-benz b!acridine-12-carboxylic acidhydrochloride; (3) treating 3-(N,N-diethylamino)ethoxy-benzb!acridine-12-carboxylic acid hydrochloride with p-toluenesulfonylchloride and benzyl 3,5-dimethyl-4-hydroxybenzoate to produce(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-(N,N-diethylamino)ethoxy-benz b!acridinium-12-carboxylate; and (4)treating (4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl3-(N,N-diethylamino)ethoxy-benz b!acridinium-12-carboxylate with methylfluorosulfonate to produce 3-QAE-LEAE-Bz; in the case of(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate (2-MeO-LEAE-Bz):(1) reactingp-anisidine with 3-hydroxy-2-naphthoic acid to produce3-(4-methoxy)anilino-2-naphthoic acid; (2) treating3-(4-methoxy)anilino-2-naphthoic acid with phosphorousoxy chloride toproduce 12-chloro-2-methoxy-benz b!acridine; (3) treating12-chloro-2-methoxy-benz b!acridine with potassium cyanide and copper(I) cyanide to produce 12-cyano-2-methoxy-benz b!acridine; (4) treating12-cyano-2-methoxy-benz b!acridine with sulfuric acid to produce2-hydroxy-benz b!acridine-12-carboxylic acid hydrosulfate; (5) treating2-hydroxy-benz b!acridine-12-carboxylic acid hydrosulfate with cesiumcarbonate and iodomethane to produce methyl 2-methoxy-benzb!acridine-12-carboxylate; (6) treating methyl 2-methoxy-benzb!acridine-12-carboxylate with sodium hydroxide in methanol andacidifying to produce 2-methoxy-benz b!acridine-12-carboxylic acid; (7)treating 2-methoxy-benz b!acridine-12-carboxylic acid withp-toluenesulfonyl chloride and benzyl 3,5-dimethyl-4-hydroxybenzoate toproduce (4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 2-methoxy-benzb!acridinium-12-carboxylate; and (8) treating(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 2-methoxy-benzb!acridinium-12-carboxylate with methyl fluorosulfonate to produce2-MeO-LEAE-Bz; in the case of(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl5-methyl-2-(trimethylammonium)ethoxy-benz b!acridinium-12-carboxylatedifluorosulfonate (2-QAE-LEAE-NHS):(1) treating 2-hydroxy-benzb!acridine-12-carboxylic acid hydrochloride with cesium carbonate andN,N-dimethylaminoethyl bromide hydrobromide to produceN,N-dimethylaminoethyl 2-(N,N-dimethylamino) ethoxy-benzb!acridine-12-carboxylate; (2) treating N,N-dimethylaminoethyl2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylate with sodiumhydroxide in methanol and acidifying to produce2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylic acid; (3)treating 3,5-dimethyl-4-hydroxybenzoic acid with N-hydroxysuccinimideand 1,3-dicyclohexylcarbodiimide to produce succinimidyl3,5-dimethyl-4-hydroxybenzoate; (4) treating2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylic acid withp-toluenesulfonyl chloride and succinimidyl3,5-dimethyl-4-hydroxybenzoate to produce(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylate; and (5)treating (2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl2-(N,N-dimethylamino)ethoxy-benz b!acridine-12-carboxylate with methylfluorosulfonate to produce 2-QAE-LEAE-NHS; in the case of(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl 5-(3-sulfopropyl)-benzb!acridinium-12-carboxylate (NSP-LEAE-Bz):(1) treating(4'-benzyloxycarbonyl-2',6'-dimethyl)phenyl benzb!acridine-12-carboxylate with 1,3-propane sultone to give NSP-LEAE-Bz;in the case of (2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl2-methoxy-5-(2-sulfoethyl)-benz b!acridinium-12-carboxylate(2-MeO-NSE-LEAE-NHS):(1) treating 2-methoxy-benzb!acridine-12-carboxylic acid with p-toluenesulfonyl chloride andsuccinimidyl 3,5-dimethyl-4-hydroxybenzoate to produce(2',6'-dimethyl-4'-succinimidyloxycarbonyl)phenyl 2-methoxy-benzb!acridinium-12-carboxylate; and (2) reacting(2',6'-dimethyl-4'-succinimidyloxycarbonyl) phenyl 2-methoxy-benzb!acridinium-12-carboxylate with ethylenesulfonyl chloride to produce2-MeO-NSE-LEAE-NHS; in the case of{2',6'-dimethyl-4'-(2-methoxyiminoethyl)}phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate dichloride (2-MeO-LEAE-Imidate):(1) treating2-methoxy-benz b!acridine-12-carboxylic acid with p-toluenesulfonylchloride, triethylamine, and 4-cyanoethyl-2,6-dimethylphenol to produce(4'-cyanoethyl-2',6'-dimethyl)phenyl 2-methoxy-benzb!acridine-12-carboxylate; (2) treating(4'-cyanoethyl-2',6'-dimethyl)phenyl 2-methoxy-benzb!acridine-12-carboxylate with methyl fluorosulfonate to produce(4'-cyanoethyl-2',6'-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate; and (3) treating(4'-cyanoethyl-2',6'-dimethyl)phenyl 2-methoxy-5-methyl-benzb!acridinium-12-carboxylate fluorosulfonate with anhydrous methanol andanhydrous hydrogen chloride to produce 2-MeO-LEAE-Imidate.
 12. A kit fordetecting and/or quantitating two or more substances in a test samplecomprising at least two different chemiluminescent compounds, eachcompound being conjugated to a binding partner which is specific for atleast one substance in a test sample, said at least two differentchemiluminescent compounds comprising (a) a linear aromatic four-ringfused acridinium compound and (b) an angular aromatic four-ring fusedacridinium compound or a three-ring acridinium compound.
 13. A test kitfor performing a simultaneous assay on two or more substances in a testsample comprising:a) a first chemiluminescent reagent comprising alinear aromatic four-ring fused acridinium compound; and (b) a secondchemiluminescent reagent comprising a three-ring acridinium compound.14. A test kit as recited in claim 13, wherein said linear aromaticfour-ring fused acridinium compound is N-alkylated.
 15. A test kit forperforming a simultaneous assay on two or more substances in a testsample comprising at least two different chemiluminescent reagents, saidreagents having discernible emission spectra for detecting and/orquantitating said test substances, said at least two differentchemiluminescent reagents comprising (a) a linear aromatic four-ringfused acridinium compound and (b) an angular aromatic four-ring fusedacridinium compound or a three-ring acridinium compound.
 16. A test kitas recited in claim 15, wherein at least one of said chemiluminescentreagents comprises a N-alkylated four-ring fused acridinium compound.17. A kit for detecting and/or quantitating two or more substances in atest sample comprising at least two different chemiluminescentcompounds, each compound being conjugated to a binding partner which isspecific for at least one substance in a test sample, said at least twodifferent chemiluminescent compounds comprising (a) an acridinium ringsystem to which an aromatic ring is fused in a linear relationship, withor without an additional fused aromatic ring, and (b) an acridinium ringsystem or an acridinium ring system to which an aromatic ring is fusedin an angular relationship, with or without an additional fused aromaticring.
 18. A test kit for performing a simultaneous assay on two or moresubstances in a test sample comprising:(a) a first chemiluminescentreagent comprising an acridinium ring system to which an aromatic ringis fused in a linear relationship, with or without an additional fusedaromatic ring; and (b) a second chemiluminescent reagent comprising anacridinium ring system.
 19. A test kit as recited in claim 18, whereinat least one of said chemiluminescent reagents is N-alkylated.
 20. Atest kit for performing a simultaneous assay on two or more substancesin a test sample comprising at least two different chemiluminescentreagents, said reagents having discernible emission spectra fordetecting and/or quantitating said test substances, said at least twodifferent chemiluminescent reagents comprising (a) an acridinium ringsystem to which an aromatic ring is fused in a linear relationship, withor without an additional fused aromatic ring, and (b) an acridinium ringsystem or an acridinium ring system to which an aromatic ring is fusedin an angular relationship, with or without an additional fused aromaticring.
 21. A test kit as recited in claim 20, wherein at least one ofsaid chemiluminescent reagents is N-alkylated.